Nucleic acids encoding modulators of body weight

ABSTRACT

The present invention relates generally to the control of body weight of animals including mammals and humans, and more particularly to nucleic acids encoding materials identified herein as modulators of weight. In its broadest aspect, the present invention relates to the elucidation and discovery of nucleotide sequences, and proteins putatively expressed by such nucleotides, that demonstrate the ability to participate in the control of mammalian body weight. The nucleotide sequences in object represent the genes corresponding to the murine and human ob gene, that have been postulated to play a critical role in the regulation of body weight and adiposity. Preliminary data, presented herein, suggests that the polypeptide product of the gene in question functions as a hormone. The present invention further provides nucleic acid molecules for use as molecular probes, or as primers for polymerase chain reaction (PCR) amplification, i.e., synthetic or natural oligonucleotides.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/635,864, filed Aug. 10, 2000, now U.S. Pat. No. 7,544,492, which hasbeen allowed, which is a continuation of U.S. application Ser. No.08/438,431, filed May 10, 1995, now U.S. Pat. No. 6,429,290, which is acontinuation-in-part of U.S. application Ser. No. 08/347,563, filed Nov.30, 1994, now U.S. Pat. No. 5,935,810, which is a continuation-in-partof U.S. application Ser. No. 08/292,345, filed Aug. 17, 1994, now U.S.Pat. No. 6,001,968, each of which is incorporated by reference in itsentirety.

The present application is a continuation-in-part of copendingapplication Ser. No. 08/347,563, filed Nov. 30, 1994, which in turn is acontinuation-in-part of copending application Ser. No. 08/292,345, filedAug. 17, 1994, of which the instant application claims the benefit ofthe filing date pursuant to 35 U.S.C. §120, and each of which isincorporated herein by reference in its entirety.

The research leading to the present inventions was funded in part byGrant No. DK 41096 from the National Institutes of Health. Thegovernment may have certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the control of body weight ofmammals including animals and humans, and more particularly to materialsidentified herein as modulators of weight, and to the diagnostic andtherapeutic uses to which such modulators may be put.

BACKGROUND OF THE INVENTION

Obesity, defined as an excess of body fat relative to lean body mass, isassociated with important psychological and medical morbidities, thelatter including hypertension, elevated blood lipids, and Type II ornon-insulin-dependent diabetes melitis (NIDDM). There are 6-10 millionindividuals with NIDDM in the U.S., including 18% of the population of65 years of age (Harris et al., 1987). Approximately 45% of males and70% of females with NIDDM are obese, and their diabetes is substantiallyimproved or eliminated by weight reduction (Harris, 1991). As describedbelow, both obesity and NIDDM are strongly heritable, though thepredisposing genes have not been identified. The molecular genetic basisof these metabolically related disorders is an important, poorlyunderstood problem.

The assimilation, storage, and utilization of nutrient energy constitutea complex homeostatic system central to survival of metazoa. Amongland-dwelling mammals, storage in adipose tissue of large quantities ofmetabolic fuel as triglycerides is crucial for surviving periods of fooddeprivation. The need to maintain a fixed level of energy stores withoutcontinual alterations in the size and shape of the organism requires theachievement of a balance between energy intake and expenditure. However,the molecular mechanisms that regulate energy balance remain to beelucidated. The isolation of molecules that transduce nutritionalinformation and control energy balance will be critical to anunderstanding of the regulation of body weight in health and disease.

An individual's level of adiposity is, to a large extent, geneticallydetermined. Examination of the concordance rates of body weight andadiposity amongst mono- and dizygous twins or adoptees and theirbiological parents have suggested that the heritability of obesity(0.4-0.8) exceeds that of many other traits commonly thought to have asubstantial genetic component, such as schizophrenia, alcoholism, andatherosclerosis (Stunkard et al., 1990). Familial similarities in ratesof energy expenditure have also been reported (Bogardus et al., 1986).Genetic analysis in geographically delimited populations has suggestedthat a relatively small number of genes may account for the 30%-50% ofvariance in body composition (Moll et al., 1991). However, none of thegenes responsible for obesity in the general population have beengenetically mapped to a definite chromosomal location.

Rodent models of obesity include seven apparently single-gene mutations.The most intensively studied mouse obesity mutations are the ob (obese)and db (diabetes) genes. When present on the same genetic strainbackground, ob and db result in indistinguishable metabolic andbehavioral phenotypes, suggesting that these genes may function in thesame physiologic pathway (Coleman, 1978). Mice homozygous for eithermutation are hyperphagic and hypometabolic, leading to an obesephenotype that is notable at one month of age. The weight of theseanimals tends to stabilize at 60-70 g (compared with 30-35 g in controlmice). ob and db animals manifest a myriad of other hormonal andmetabolic changes that have made it difficult to identify the primarydefect attributable to the mutation (Bray et al., 1989).

Each of the rodent obesity models is accompanied by alterations incarbohydrate metabolism resembling those in Type II diabetes in man. Insome cases, the severity of the diabetes depends in part on thebackground mouse strain (Leiter, 1989). For both ob and db, congenicC57BL/Ks mice develop a severe diabetes with ultimate β cell necrosisand islet atrophy, resulting in a relative insulinopenia. Conversely,congenic C57BL/6J ob and db mice develop a transient insulin-resistantdiabetes that is eventually compensated by cell hypertrophy resemblinghuman Type II diabetes.

The phenotype of ob and db mice resembles human obesity in ways otherthan the development of diabetes—the mutant mice eat more and expendless energy than do lean controls (as do obese humans). This phenotypeis also quite similar to that seen in animals with lesions of theventromedial hypothalamus, which suggests that both mutations mayinterfere with the ability to properly integrate or respond tonutritional information within the central nervous system. Support forthis hypothesis comes from the results of parabiosis experiments(Coleman, 1973) that suggest ob mice are deficient in a circulatingsatiety factor and that db mice are resistant to the effects of the obfactor (possibly due to an ob receptor defect). These experiments haveled to the conclusion that obesity in these mutant mice may result fromdifferent defects in an afferent loop and/or integrative center of thepostulated feedback mechanism that controls body composition.

Using molecular and classical genetic markers, the ob and db genes havebeen mapped to proximal chromosome 6 and midchromosome 4, respectively(Bahary et al., 1990; Friedman et al., 1991b). In both cases, themutations map to regions of the mouse genome that are syntonic withhuman, suggesting that, if there are human homologs of ob and db, theyare likely to map, respectively, to human chromosomes 7q and 1p. Defectsin the db gene may result in obesity in other mammalian species: ingenetic crosses between Zucker fa/fa rats and Brown Norway +/+rats, thefa mutation (rat chromosome 5) is flanked by the same loci that flank dbin mouse (Truett et al., 1991).

Because of the myriad factors that seem to impact body weight, it isdifficult to speculate as to which of these factors, and moreparticularly, which homeostatic mechanism is actually primarilydeterminative. Nonetheless, the apparent connection between the ob geneand the extent and characteristics of obesity have prompted the furtherinvestigation and elucidation that is reflected by the presentapplication. It is the identification of the sequence of the gene andcorresponding peptide materials, to which the present inventionfollowing below directs itself.

The citation of any reference herein should not be construed as anadmission that such reference is prior art to the instant invention.Full citations of references cited by author and year are found at theend of the specification.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to the elucidationand discovery of nucleotide sequences, and proteins putatively expressedby such nucleic acids or degenerate variations thereof, that demonstratethe ability to participate in the control of mammalian body weight. Thenucleotide sequences in object are believed to represent the genescorresponding to the murine and human ob gene, that is postulated toplay a critical role in the regulation of body weight and adiposity.Data presented herein indicates that the polypeptide product of the genein question is secreted by the cells that express it and that thepolypeptide functions as a hormone.

In addition, the Examples herein demonstrate that the ob polypeptide,alternatively termed herein “leptin,” circulates in mouse, rat, andhuman plasma. Leptin is absent in plasma from ob/ob mice, and is presentat ten-fold higher concentrations in plasma from db/db mice, andtwenty-fold higher concentrations in fa/fa rats. Most significantly,daily injections of recombinant leptin dramatically reduces the bodymass of ob/ob mice, significantly effects the body weight of wild-typemice, and has no effect on db/db mice.

In a further aspect, the ob polypeptide from one species is biologicallyactive in another species. In particular, the human ob polypeptide isactive in mice.

In a first instance, the modulators of the present invention comprisenucleic acid molecules, including recombinant DNA molecules (e.g., cDNAor a vector containing the cDNA or isolated genomic DNA) or cloned genes(i.e. isolated genomic DNA), or degenerate variants thereof, whichencode polypeptides themselves serving as modulators of weight controlas hereinafter defined, or conserved variants or fragments thereof,particularly such fragments lacking the signal peptide (alternativelyreferred to herein as mature ob polypeptide), which polypeptides possessamino acid sequences such as set forth in FIG. 1 (SEQ ID NO:2), FIG. 3(SEQ ID NO:4), FIG. 5 (SEQ ID NO:5) and FIG. 6 (SEQ ID NO:6). Inspecific embodiments, amino acid sequences for two variants of murineand human ob polypeptides are provided. Both polypeptides are found in aform with glutamine 49 deleted, which may result from an mRNA splicinganomaly. The ob polypeptides from various species may be highlyhomologous; as shown in FIG. 4, murine and human ob polypeptides aregreater than 80% homologous.

The nucleic acid molecules, recombinant DNA molecules, or cloned genes,may have the nucleotide sequences or may be complementary to DNA codingsequences shown in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3). Inparticular, such DNA molecules can be cDNA or genomic DNA isolated fromthe chromosome. Nucleic acid molecules of the invention may alsocorrespond to 5′ and 3′ flanking sequences of the DNA. Accordingly, thepresent invention also relates to the identification of a gene having anucleotide sequence selected from the sequences of FIG. 1 (SEQ ID NO:1)and FIG. 2 (SEQ ID NO:3) herein, and degenerate variants, allelicvariations, and like cognate molecules.

A nucleic acid molecule of the invention can be DNA or RNA, includingsynthetic variants thereof having phosphate or phosphate analog, e.g.,thiophosphate, bonds. Both single stranded and double stranded sequencesare contemplated herein.

The present invention further provides nucleic acid molecules for use asmolecular probes, or as primers for polymerase chain reaction (PCR)amplification, i.e., synthetic or natural oligonucleotides having asequence corresponding to a portion of the sequences shown in FIG. 1(SEQ ID NO:1), FIG. 2 (SEQ ID NO:3) and FIG. 20A (SEQ ID NO:22), or the5′ and 3′ flanking sequences of the coding sequences. In particular, theinvention contemplates a nucleic acid molecule having at least about 10nucleotides, wherein a sequence of the nucleic acid molecule correspondsto a nucleotide sequence of the same number of nucleotides in thenucleotide sequences of FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:3) andFIG. 20A (SEQ ID NO:22), or a sequence complementary thereto. Morepreferably, the nucleic acid sequence of the molecule has at least 15nucleotides. Most preferably, the nucleic acid sequence has at least 20nucleotides. In an embodiment of the invention in which theoligonucleotide is a probe, the oligonucleotide is detectably labeled,e.g., with a radionuclide (such as ³²P), or an enzyme.

In further aspects, the present invention provides a cloning vector,which comprises the nucleic acids of the invention that encode the obpolypeptide; and a bacterial, insect, or a mammalian expression vector,which comprises the nucleic acid molecules of the invention encoding theob polypeptide, operatively associated with an expression controlsequence. Accordingly, the invention further relates to a host cell,such as a bacterial cell, yeast cell, insect cell, or a mammalian cell,transfected or transformed with an appropriate expression vector, andcorrespondingly, to the use of the above mentioned constructs in thepreparation of the modulators of the invention.

In yet a further aspect, the present invention relates to antibodiesthat bind to the ob polypeptide. Such antibodies may be generatedagainst the full length polypeptide, or antigenic fragments thereof. Inone aspect, such antibodies inhibit the functional (i.e., body weightand fat composition modulating) activity of the ob polypeptide. Inanother aspect, antibodies can be used to determine the level ofcirculating ob polypeptide in plasma or serum. In yet a further aspect,regio-specific antibodies, particularly monoclonal antibodies, can beused as probes of ob polypeptide structure.

All of the foregoing materials are to be considered herein as modulatorsof body weight and fat composition, and as such, may be used in avariety of contexts. Specifically, the invention contemplates bothdiagnostic and therapeutic applications, as well as certain agriculturalapplications, all contingent upon the use of the modulators definedherein, including both nucleic acid molecules and peptides. Moreover,the modulation of body weight carries specific therapeutic implicationsand benefits, in that conditions where either obesity or, conversely,cachexia represent undesired bodily conditions, can be remedied by theadministration of one or more of the modulators of the presentinvention.

Thus, a method for modulating body weight of a mammal is proposed thatcomprises controlling the expression of the protein encoded by a nucleicacid having nucleotide sequence selected from the sequence of FIG. 1(SEQ ID NO:1), the sequence of FIG. 2 (SEQ ID NO:3) and degenerate andallelic variants thereof. Such control may be effected by theintroduction of the nucleotides in question by gene therapy into fatcells of the patient or host to control or reduce obesity. Conversely,the preparation and administration of antagonists to the nucleotides,such as anti-sense molecules, would be indicated and pursued in theinstance where conditions involving excessive weight loss, such asanorexia nervosa, cancer, or AIDS are present and under treatment. Suchconstructs would be introduced in similar fashion to the nucleotides,directly into fat cells to effect such changes.

Correspondingly, the proteins defined by FIGS. 1, 3, 5, and 6 (SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6), conserved variants,active fragments thereof, and cognate small molecules could beformulated for direct administration for therapeutic purposes, to effectreduction or control of excessive body fat or weight gain.Correspondingly, antibodies and other antagonists to the stated proteinmaterials, such as fragments thereof, could be prepared and similarlyadministered to achieve the converse effect. Accordingly, the inventionis advantageously directed to a pharmaceutical composition comprising anob polypeptide of the invention, or alternatively an antagonist thereof,in an admixture with a pharmaceutically acceptable carrier or excipient.

The diagnostic uses of the present nucleotides and correspondingpeptides extend to the use of the nucleotides to identify furthermutations of allelic variations thereof, so as to develop a repertoireof active nucleotide materials useful in both diagnostic and therapeuticapplications. In particular, both homozygous and heterozygous mutationsof the nucleotides in question could be prepared that would bepostulated to more precisely quantitate the condition of patients, todetermine the at-risk potential of individuals with regard to obesity.Specifically, heterozygous mutations are presently viewed as associatedwith mild to moderate obesity, while homozygous mutations would beassociated with a more pronounced and severe obese condition.Corresponding DNA testing could then be conducted utilizing theaforementioned ascertained materials as benchmarks, to facilitate anaccurate long term prognosis for particular tendencies, so as to be ableto prescribe changes in either dietary or other personal habits, ordirect therapeutic intervention, to avert such conditions.

The diagnostic utility of the present invention extends to methods formeasuring the presence and extent of the modulators of the invention incellular samples or biological extracts (or samples) taken from testsubjects, so that both the encoded nucleotide (genomic DNA or RNA) andor the levels of protein in such test samples could be ascertained.Given that the increased activity of the nucleotide and presence of theresulting protein reflect the capability of the subject to inhibitobesity, the physician reviewing such results in an obese subject woulddetermine that a factor other than dysfunction with respect to thepresence and activity of the nucleotides of the present invention is acause of the obese condition. Conversely, depressed levels of thenucleotide and or the expressed protein would suggest that such levelsmust be increased to treat such obese condition, and an appropriatetherapeutic regimen could then be implemented.

Further, the nucleotides discovered and presented in FIGS. 1 and 2represent cDNA in which, as stated briefly above, is useful in themeasurement of corresponding RNA. Likewise, recombinant protein materialcorresponding to the polypeptides of FIGS. 1 and 3 may be prepared andappropriately labeled, for use, for example, in radioimmunoassays, forexample, for the purpose of measuring fat and/or plasma levels of the obprotein, or for detecting the presence and level of a receptor for ob ontissues, such as the hypothalamus.

Yet further, the present invention contemplates not only theidentification of the nucleotides and corresponding proteins presentedherein, but the elucidation of the receptor to such materials. In suchcontext, the polypeptides of FIGS. 1, 3, 5, and/or 6 could be preparedand utilized to screen an appropriate expression library to isolateactive receptors. The receptor could thereafter be cloned, and thereceptor alone or in conjunction with the ligand could thereafter beutilized to screen for small molecules that may possess like activity tothe modulators herein.

Yet further, the present invention relates to pharmaceuticalcompositions that include certain of the modulators hereof, preferablythe polypeptides whose sequences are presented in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:5 and SEQ ID NO:6, their antibodies, corresponding smallmolecule agonists or antagonists thereof, or active fragments preparedin formulations for a variety of modes of administration, where suchtherapy is appropriate. Such formulations would include pharmaceuticallyacceptable carriers, or other adjuvants as needed, and would be preparedin effective dosage ranges to be determined by the clinician or thephysician in each instance.

Accordingly, it is a principal object of the present invention toprovide modulators of body weight as defined herein in purified form,that exhibit certain characteristics and activities associated withcontrol and variation of adiposity and fat content of mammals.

It is a further object of the present invention to provide methods forthe detection and measurement of the modulators of weight control as setforth herein, as a means of the effective diagnosis and monitoring ofpathological conditions wherein the variation in level of suchmodulators is or may be a characterizing feature.

It is a still further object of the present invention to provide amethod and associated assay system for the screening of substances, suchas drugs, agents and the like, that are potentially effective to eithermimic or inhibit the activity of the modulators of the invention inmammals.

It is a still further object of the present invention to provide amethod for the treatment of mammals to control body weight and fatcontent in mammals, and or to treat certain of the pathologicalconditions of which abnormal depression or elevation of body weight is acharacterizing feature.

It is a still further object of the present invention to prepare geneticconstructs for use in genetic therapeutic protocols and orpharmaceutical compositions for comparable therapeutic methods, whichcomprise or are based upon one or more of the modulators, bindingpartners, or agents that may control their production, or that may mimicor antagonize their activities.

Other objects and advantages will become apparent to those skilled inthe art from a review of the ensuing description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleic acid sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:2) derived for the murine ob cDNA. A 97 basepair 5′ leader was followed by a predicted 167 amino acid open readingframe and an approximately 3700 kB 3′ untranslated sequence. A total ofabout 2500 base pairs of the 3′ untranslated sequence is shown. Analysisof the predicted protein sequence by observation and using the SigSeqcomputer program indicates the presence of a signal sequence(underlined). Microheterogeneity of the cDNA was noted in thatapproximately 70% of the cDNAs had a glutamine codon at codon 49 and 30%did not (see FIGS. 5 and 6, infra). This amino acid is underlined, as isthe arginine codon that is mutated in C57BL/6J ob/ob mice (1J mice).

FIG. 2 depicts the nucleic acid sequence (SEQ ID NO:3) derived for thehuman ob cDNA. The nucleotides are numbered from 1 to 701 with a startsite at nucleotide 46 and a termination at nucleotide 550.

FIG. 3 depicts the full deduced amino acid sequence (SEQ ID NO:4)derived for the human ob gene corresponding to the nucleic acid sequenceof FIG. 2. The amino acids are numbered from 1 to 167. A signal sequencecleavage site is located after amino acid 21 (Ala) so that the matureprotein extends from amino acid 22 (Val) to amino acid 167 (Cys).

FIG. 4 depicts the comparison between the murine (SEQ ID NO:2) and human(SEQ ID NO:4) deduced amino acid sequences. The sequence of the human obdeduced amino acid sequence was highly homologous to that of mouse.Conservative changes are noted by a dash, and non-conservative changesby an asterisk. The variable glutamine codon is underlined, as is theposition of the nonsense mutation in C57BL/6J ob/ob (1J) mice. Overall,there is 84% identity at the amino acid level, although only sixsubstitutions were found between the valine at codon 22 (immediatelydownstream of the signal sequence overage) and the cysteine at position117.

FIG. 5 depicts the full length amino acid sequence (SEQ ID NO:5) derivedfor the murine ob gene as shown in FIG. 3, but lacking glutamine atposition 49. The nucleotides are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 6 depicts the full deduced amino acid sequence (SEQ ID NO:6)derived for the human ob gene as shown in FIG. 4, but lacking glutamineat position 49. The nucleotides are numbered from 1 to 166. A signalsequence cleavage site is located after amino acid 21 (Ala) (and thus,before the glutamine 49 deletion) so that the mature protein extendsfrom amino acid 22 (Val) to amino acid 166 (Cys).

FIG. 7. (A) Physical map of the location of ob in the murine chromosome,and the YAC and P1 cloning maps. “M and N” corresponds to MulI and NotIrestriction sites. The numbers correspond to individual animals thatwere recombinant in the region of ob of the 1606 meioses that werescored. Met, Pax 4, D6Rck39, D6Rck13, and Cpa refer to locations in theregion of ob that bind to the DNA probes. YACs were isolated usingD6Rck13 and Pax-4 as probes, and the ends were recovered usingvectorette PCR and/or plasmid end rescue and used in turn to isolate newYACs. (B) The resulting YAC contig. One of the YACs in this contig,Y902A0925, was chimeric. Each of the probes used to genotype therecombinant animals is indicated in parentheses. (6) Corresponds to YAC107; (5) corresponds to M16(+) (or M16(pLUS)); (4) corresponds toadu(+); (3) corresponds to aad(pICL); (2) corresponds to 53(pICL); and(1) corresponds to 53(+). (C) The P1 contig of bacteriophage P1 clonesisolated with selected YAC end probes. The ob gene was isolated in a P1clone isolated using the distal end of YAC YB6S2F12 (end (4))(alternatively termed herein adu(+)).

FIG. 8 presents a photograph of an ethidium bromide stain of 192independent isolates of the fourth exon trapping experiment that werePCR amplified and characterized.

FIG. 9 is a photograph of an ethidium bromide stain of PCR-amplifiedclones suspected of carrying ob. Each of the 7 clones that did not carrythe artifact was reamplified using PCR and electrophoresed on a 1%agarose gel in TBE and stained with ethidium bromide. The size markers(far left unnumbered lane) are the commercially available “1 kB ladder”.Lane 1—clone 1D12, containing an “HIV sequence.” Lane 2—clone 1F1, anovel clone outside of the ob region. Lane 3—clone 1H3. Lane 4—clone2B2, which is the identical to 1F1. Lane 5—clone 2G7, which contains anob exon. Lane 6—clone 2G11, which is identical to 1F1. Lane 7—clone 2H1,which does not contain an insert.

FIG. 10 presents the sequence of the 2G7 clone (SEQ ID NO:7), whichincludes an exon coding for a part of the ob gene. The primer sequencesused to amplify this exon are boxed in the figure (SEQ ID NOS:8 and 9).

FIG. 11. (A) Reverse transcription-PCR analysis of mRNA from differenttissues of the same mouse with the 2G7 primers and actin primers. TheRT-PCR reactions were performed using 100 ng of total RNA reversetranscribed with oligo dT as a primer for first strand cDNA. PCRamplification was performed for 35 cycles with 94° denaturation×1′; 55°hybridization×1′; and 72 extensions for 2′ with a 1′ secondautoextension per cycle. RT-PCR products were resolved in a 2% lowmelting point agarose gel run in 1×TBE buffer. (B) Northern blot of mRNAfrom different organs of the mouse using PCR labeled 2G7 as a probe. Tenμg of total RNA from each of the tissues was electrophoresed on anagarose gel with formaldehyde. The probe was hybridized at 65° C. inRapid Hybe (Amersham). Autoradiographic signals were apparent after 1hour of exposure; the experiment shown was the result of a 24 hourexposure.

FIG. 12. (A) An ethidium bromide stain from an RT PCR reaction on fatcell (white adipose tissue) RNA from each of the mouse strains listed.Total RNA (100 ng) for each sample was reverse transcribed using oligodT and reverse transcriptase, and the resulting single stranded cDNA wasPCR amplified with the 2G7 primers (lower bands) or actin primers (upperbands). Both the 2G7 and actin primers were included in the same PCRreaction. The products were run on a 1% agarose TBE gel. (B) Northernanalysis corresponding to (A). Ten μg of fat cell (white adipose tissue)RNA from each of the strains indicated were run out and probed with thePCR labeled 2G7 probe as in FIG. 11B, above. An approximately 20-foldincrease in the level of 2G7 mRNA was apparent in white fate RNA fromthe C57BL/6J ob/ob (1J) strain relative to lean littermates. In both theRT-PCR and Northern experiments there was no detectable signal in 2G7RNA from the SM/Ckc−+^(Dac)ob^(2J)/ob^(2J) (2J) mice even after a 2 weekexposure. A 24 hour autoradiographic exposure is shown. The same filterwas hybridized to an actin probe (bottom portion of the panel).

FIG. 13 is a Northern analysis of additional 2J animals and controlanimals that confirms the absence of the ob mRNA from 2J animals. TheNorthern analysis was performed as in FIGS. 11 and 12. In this case, thecontrol RNA was ap2, a fat specific transcript. There is no significanceto the varying density of the ap2 bands.

FIG. 14 compares the DNA sequence of the C57BL/6J (normal) and theC57BL/6J ob/ob (1J) mice in the region of the point mutation that leadsto introduction of a premature stop codon (nonsense mutation) in themutant strain cDNA. The ob/ob mice had a C→T mutation that changed anarginine residue at position 105. This base change is shown as theoutput from the automated DNA sequencer. RT-PCR was performed usingwhite fat RNA from both strains (+/+ and ob/ob) using primers from the5′ and 3′ untranslated regions. The PCR reaction products were gelpurified and directly sequenced manually and using an ABI 373A automatedsequencer with primers along both strands of the coding sequence.

FIG. 15. (A) Genomic southern blot of genomic DNA from each of the mousestrains listed. Approximately 5 μg of DNA (derived from genomic DNAprepared from liver, kidney or spleen) was restriction digested with therestriction enzyme indicated. The DNA was then electrophoresed in a 1%agarose TBE gel and probed with PCR labeled 2G7. Restriction digestionwith BglII revealed an increase in the size of an approximately 9 kB(the largest) BglII fragment in SM/Ckc−+Dacob^(2J)/ob^(2J) (2J) DNA.RFLPs were not detectable with any other restriction enzymes.Preliminary restriction mapping of genomic DNA indicated that thepolymorphic BglII site is about 7 kB upstream of the transcription startsite. None of the other enzymes tested extend past the mRNA start site.(B) Segregation of a BglII polymorphism in theSM/Ckc−+Dacob^(2J)/ob^(2J) strain. Six obese and five lean progeny fromthe same generation of the coisogenic SM/Ckc−+^(Dac)ob^(2J)/ob^(2J) (2J)colony were genotyped by scoring the BglII polymorphism as shown in (A).All of the phenotypically obese animals were homozygous for the largerallele of the polymorphic Bgl fragment. The DNA in the “control” lanewas prepared from an unrelated SM/Ckc−+^(Dac)+/+ mouse, bred separatelyfrom the SM/Ckc−+^(Dac)ob^(2J)/ob^(2J) colony.

FIG. 16 is a Southern blot of EcoRI digested genomic DNA from thespecies listed, using an ob cDNA as a probe (i.e., a zoo blot).Hybridization signals were detectable in every vertebrate sample, evenafter a moderate stringency hybridization. The cat DNA in thisexperiment was slightly degraded. The restricted DNA was run on a 1%agarose TBE gel, and transferred to an imobilon membrane for probing.The filter was hybridized at 65° C. and washed in 2×SSC/0.2% SDS at 65°C. twice for twenty minutes and exposed for 3 days using Kodak X-OMATfilm.

FIG. 17 presents the expression cloning region of vector pET-15b(Novagen).

FIG. 18 presents analysis of the eluate from a His-binding resin (Ni)column for a recombinant mature murine ob fusion to a His-tag (A) andmature human ob fusion to a His-tag (B). Bacteria transformed withvectors pETM9 and pETH14, respectively. Upon induction with 1 mM IPTG atoptimal conditions, the transformed bacteria were able to produce100-300 μg/ml of ob fusion protein, primarily in the inclusion body. Theinclusion body was solubilized with 6M guanidine-HCl or urea, and fusionprotein (present in the lysis supernatant) was loaded on the His-bindingresin (Ni) column in 10 ml of 1× binding buffer with urea. The columnwas eluted stepwise with 5 ml aliquots of 20 μM, 60 μM, and 300 μMimidazole, and finally with strip buffer. The aliquots were analyzed forthe presence of ob polypeptide fusion on a 15% acrylamide gel. Each lanecontains the equivalent of 100 μl of bacterial extract.

FIG. 19. (A) In vitro translation of ob RNA. A human ob cDNA wassubcloned into the pGEM vector. The plasmid was linearized and plusstrand RNA was synthesized using sp6 polymerase. The in vitrosynthesized RNA was translated in the presence or absence of caninepancreatic microsomal membranes. An approximately 18 kD primarytranslation product was seen after in vitro translation. The addition ofmicrosomal membranes to the reaction led to the appearance of a secondtranslation product about 2 kD smaller than the primary translationproduct. The size of the translation product of interleukin-1α RNA,which lacks an encoded signal sequence, was unchanged by the addition ofmicrosomal membranes. These data indicated the presence of a functionalsignal sequence. (B) In vitro translation in the presence or absence ofproteinase K. Protease treatment resulted in complete proteolysis of the18 kD primary translation product, while the 16 kD processed form wasunaffected. Permeabilization of the microsome with 0.1% TRITON-X100rendered the processed from protease sensitive. These results indicatethat the product had translated into the lumen of the microsome.

FIG. 20. (A) The sequence of the human ob gene (SEQ ID NO:22). (B) Aschematic diagram of the murine ob gene. (C) A schematic diagram of thehuman ob gene. In both (B) and (C), the start and stop codons areunderlined. There is no evidence of a first intron homologous to themouse first intron in the human gene, but its existence cannot beexcluded.

FIG. 21 presents a schematic drawing of one of the cloning strategiesemployed to achieve recombinant expression of ob in pichia yeast. (A)Expression vector of ob with an α-mating factor signal sequence. (B)Schematic drawing of the structure of the recombinant fusion protein,including the amino acid sequence (SEQ ID NO:23) showing the XhoI siteand putative KEX-2 and STE-13 cleavage sites, and the N-terminal surplusamino acids present after KEX-2 cleavage (SEQ ID NO:24). (C) Analternative strategy for producing mature ob under involves preparing aconstruct with an amino acid sequence corresponding to a XhoI cleavagesite and a KEX-2 cleavage site immediately upstream of the mature obpolypeptide sequence (SEQ ID NO:25).

FIG. 22 Alternative expression strategy in pichia. (A) Expression vectorof an ob fusion with a His tag adopted from the pET expression systemunder control of the α-mating factor signal sequence. (B) Schematicdrawing of the structure of the recombinant ob fusion protein containinga His tag, which includes the α-mating factor signal sequence, putativeKEX-2 and STE-13 cleavage sites, the His-tag, and a thrombin cleavagesite, and which would yield ob with three surplus N-terminal amino acidresidues.

FIG. 23. (A) PAGE analysis of expression of murine ob (both themicroheterogenous forms, i.e., containing and missing Gln 49) intransformed pichia yeast. The expected band of approximately 16 kD isvisible in the transformed yeast culture fluid (second and third lanes),but not in culture fluid from non-transformed yeast (first lane). (B)PAGE analysis of partially purified recombinant ob polypeptide oncarboxymethyl cellulose, a weak cation exchanger. A band of about 16 kDis very visible in fractions 3 and 4 from the column, which was elutedwith 250 mM NaCl. Lane 1—loaded sample; lane 2—flow through; lanes3-5—fractions eluted with 250 mM NaCl.

FIG. 24 The ob Protein Circulates in Mouse Plasma

FIG. 24A. Immunoprecipitations from Mouse Blood

0.5 ml of mouse plasma was pre-cleared with unconjugated sepharose andincubated overnight with immunopurified anti-ob antibodies conjugated tosepharose 4B beads. The immunoprecipitate was separated on a 15%SDS-PAGE gel, transferred and Western blotted with an anti-ob antibody.The protein migrated with a molecular weight of ˜16 kD, to the sameposition as the mature mouse ob protein expressed in yeast. The proteinwas absent in plasma from C57BL/6J ob/ob mice and increased ten-fold inplasma from C57BLB/Ks db/db mice relative to wild type mice. db micehave been suggested to overproduce the ob protein, secondary toresistance to its effects.

FIG. 24B. Increased Levels of ob in Fatty Rats

The fatty rat is obese as a result of a recessive mutation on ratchromosome 5. Genetic data has suggested a defect in the same gene as ismutant in db mice. Plasma from fatty rats and lean littermates wasimmunoprecipitated and run on Western blots. A twenty-fold increase inthe circulating level of ob is seen in the mutant animals.

FIG. 24C. Quantitation of the ob Protein in Mouse Plasma

Increasing amounts of the recombinant mouse protein were added to 100λof plasma from ob mice and immunoprecipitated. The signal intensity onWestern blots was compared to that from 100λ of plasma from wild typemice. A linear increase in signal intensity was seen with increasingamounts of recombinant protein demonstrating that theimmunoprecipitations were performed under conditions of antibody excess.Similar signals were seen in the wild type plasma sample and the samplewith 2 ng of recombinant protein indicating the circulating level inmouse plasma is ˜20 ng/ml.

FIG. 24D. ob Protein in Adipose Tissue Extracts

Cytoplasmic extracts of mouse adipose tissue were prepared from db andwild type mice. Western blots showed increased levels of the 16 kDprotein in extracts prepared from db mice.

FIG. 25 The ob Protein Circulates at Variable Levels in Human Plasma

FIG. 25A. Western Blots of Human Plasma

Plasma samples were obtained from six lean volunteers.Immunoprecipitation and Western blotting revealed the presence of animmunoreactive 16 kD protein, identical in size to a recombinant 146amino acid human protein expressed in yeast. Variable levels of theprotein were seen in each of the six samples.

FIG. 25B. An ELISA (Enzyme Linked Immunoassay) for Human ob

Microtiter plates were coated with immunopurified anti-human obantibodies. Known amounts of recombinant protein were added to theplates and detected using immunopurified biotinylated anti-obantibodies. The resulting standard curve showed that the assay wascapable of detecting 1 ng/ml or more of the human ob protein.

FIG. 25C. Quantitation of the ob Protein in Human Plasma

An ELISA immunoassay was performed using 100λ of plasma from the sixlean volunteers and the standards used in panel B. Levels of the obprotein ranging from 2 ng/ml in HP1 to 15 ng/ml in HP6 were seen. Thesedata correlated with the Western blot data in panel A.

FIG. 26 The ob Protein Forms Inter- or Intramolecular Disulphide Bonds

FIG. 26A. Western blots Under Non Reducing Conditions

The Western blots of mouse and human plasma were repeated with andwithout the addition of reducing agents to the sample buffer. Whenβ-Mercaptoethanol is omitted from the sample buffer, immunoprecipitatesfrom db plasma migrate with an apparent molecular mass of 16 kD and 32kD. Addition of β-Mercaptoethanol to the buffer leads to thedisappearance of the 32 kD moiety (see FIG. 1). This result isrecapitulated when the mouse protein is expressed in the yeast, Pichiapastoris. In this case, the mouse ob protein migrates to the position ofa dimer. Under reducing conditions the purified recombinant mouseprotein migrates with an apparent molecular weight of 16 kD indicatingthat the 32 kD molecular form is the result of one or two intermoleculardisulphide bonds. The human protein expressed in vivo and in Pichiapastoris migrates with a molecular mass of 16 kD under both conditions(data not shown).

FIG. 26B. The Human Protein Expressed in Yeast Contains anIntramolecular Disulphide Bond

Secreted proteins generally assume their correct conformation whenexpressed in the Pichia pastoris expression system. The 146 amino acidmature human protein was expressed in Pichia pastoris and purified fromthe yeast media by a two-step purification protocol involving IMAC andgel filtration. The purified recombinant protein was subjected to massspectrometry before and after cyanogen bromide cleavage. Cyanogenbromide cleaves at the carboxy terminus of methionine residues. Themolecular mass of the recombinant yeast protein was 16,024±3 Da(calculated molecular mass=16,024 Da). Cyanogen bromide cleaves afterthe three methionines in the protein sequence at amino acids 75, 89 and157. The cyanogen bromide fragment with measured mass 8435.6 Dacorresponds to amino acids 90-157 and 158-167 joined by a disulphidelinkage between cys-117 and cys-167 (calculated molecular mass=8434.5Da).

FIG. 27 Preparation of Bioactive Recombinant Protein

The nucleotide sequence corresponding to the 145 amino acid mature mouseob protein was cloned into the PET 15b expression vector. This PETvector inserts a polyhistidine tract (His-tag) upstream of the clonedsequence which allows efficient purification using Immobilized MetalAffinity Chromatography (IMAC). The recombinant bacterial proteininitially partitioned in the insoluble membrane fraction after bacteriallysis. The membrane fraction was solubilized using guanidiumhydrochloride and loaded onto an IMAC column. The protein was elutedstepwise with increasing concentrations of imidazole as shown. Theeluted protein was refolded and treated with thrombin to remove theHis-tag, as described below. The final yield of soluble protein was 45ng/ml of bacterial culture.

FIG. 28 Biologic Effects of the ob Protein

FIG. 28A. Time Course of Food Intake and Body Weight

Groups of ten animals received either daily intraperitoneal injectionsof the ob protein at a dose of 5 μg/kg/day, daily injections of PBS orno treatment. The treatment groups included C57Bl/6J ob/ob mice (leftpanels), C57Bl/Ks db/db mice (center panels) and CBA/J+/+ mice (rightpanels). The food intake of the mice was measured daily and the bodyweight was recorded at three to four day intervals as indicated. (Thescale of the body weight in grams is different for the wild type micevs. the ob and db mice.) The food intake of the ob mice receivingprotein was reduced after the first injection and stabilized after thefourth day at a level ˜40% of that seen in the sham injected group(p<0.001). The body weight of these animals decreased an average of 1.3grams/day and stabilized after three weeks to a level ˜60% of thestarting weight (p<0.0001). No effect of the protein was demonstrable indb mice. Small but significant effects on body weight were observed inCBA/J mice at two early time points (p<0.02). The standard error of eachmeasure is depicted by a bar and the statistical significance of theseresults is shown in Table 1.

FIG. 28B. Pair Feeding of ob Mice

A group of four C57Bl/6J ob/ob mice were fed an amount of food equal tothat consumed by the group of ob mice receiving recombinant protein. Theweight loss for both groups was calculated after five, eight and twelvedays. The food restricted mice loss less weight than the ob micereceiving protein (p<0.02). This result indicates that the weightreducing effect of the ob protein is the result of effects on both foodintake and energy expenditure.

FIG. 28C. Photograph of a Treated ob Mouse

Shown are two C57Bl/6J ob/ob mice. The mouse on the left received PBSand weighed 65 grams which was the starting weight. The mouse on theright received daily injections of the recombinant ob protein. Thestarting weight of this animal was also 65 grams, and the weight afterthree weeks of protein treatment was 38 grams.

FIG. 28D. Livers From Treated and Untreated ob Mice

Shown are livers from treated and untreated C57Bl/6J ob/ob mice. Theliver from the mouse receiving PBS had the gross appearance of a fattyliver and weighed 5.04 grams. The liver from the mouse receiving therecombinant ob protein had a normal appearance and weighed 2.23 grams.

FIG. 29 InSitu Hybridization of ob to Adipose Tissue

Sense and Antisense ob RNA was labeled in vitro using Sp6 and t7polymerase and digoxigenin. The labeled RNAs were hybridized to paraffinembedded sections of adipose tissue from epididymal fat pads of eightweek old C57Bl/Ks mice (labelled wild type) and C57Bl/Ks db/db mice(labelled db). In the figure, the lipid droplets appear as unstainedvacuoles within cells. The cytoplasm is a thin rim at the periphery ofthe cells and is indistinguishable from the cell membrane X 65.Hybridization to all the adipocytes in the field was detected in thewild type sections only using the antisense probe and greatly increasedlevels were seen in the tissue sections from the db/db animals.

FIG. 30 ob RNA Is Expressed in Adipocytes in vivo and in vitro

Total RNA (10 micrograms) from several different sources waselectophoresed on Northern blots and hybridized to an ob probe. Firstly,differences in cell buoyancy after collagenase digestion was used topurify adipocytes. ob RNA was present only in the adipocyte fraction.(lane SV indicates the stromovascular fraction and A indicates theadipocyte fraction) in addition, ob RNA was not expressed in theundifferentiated 3T3-442 preadipocyte cells. (labelled U) Differentiatedadipocytes from these cell lines expressed clearly detectable levels ofob mRNA (labelled D).

FIG. 31 ob RNA is Expressed in All Adipose Tissue Depots

All of the adipose tissue depots tested expressed ob RNA. The inguinalfat pad expressed somewhat lower RNA levels although there wasvariability in the levels of signals in different experiments. (FIG.31A) Lanes 1) epididymal 2) inguinal 3) abdominal 4) parametrial fatpads. Brown fat also expressed a low level of ob RNA. (FIG. 31B) Thelevel of ob expression in brown fat was unchanged in animals housed at4° C. for one week while the abundance of the brown fat specific UCPRNA, known to be cold inducible, increased five-fold.

FIG. 32 Expression of ob RNA in db/db and Gold ThioGlucose Treated Mice

Total RNA from the parametrial fat pads of db/db and Gold Thioglucose(GTG) treated mice was electrophoresed on a Northern blot. GTGadministered as a single dose is known to cause obesity by inducingspecific hypothalamic lesions. One month old CBA female mice weretreated with GTG (0.2 mg/g) with a resulting increase of >20 g intreated animals relative to control animals (<5 g). Hybridization of anob probe to RNA from db/db and GTG treated mice revealed a twenty-foldincrease in the abundance of ob RNA relative to control RNA (actin orGAPDH).

FIG. 33 Northern blot analysis of human RNA. Northern blots containing10 μg of total RNA from human adipose tissue (FAT, panel A) and 2 μg ofpolyA+ RNA from other human tissues (panel B) were hybridized to humanOB or human β actin probes as indicated. An intense signal at ˜4.5 kbwas seen with the adipose tissue total RNA. Hybridization to the polyA+RNA revealed detectable signals in heart (HE) and placenta (PL), whereasOB RNA was not detected in brain (BR), lung (LU), liver (LI), skeletalmuscle (SM), kidney (KI), and pancreas (PA). In each case, the length ofthe autoradiographic exposure is indicated. Of note, the genesis of thelower molecular bands seen in placental RNA (e.g., alternate splicing,RNA degradation) is not known.

FIG. 34 YAC contig containing the human OB gene and 8 microsatellitemarkers. The YAC-based STS-content map of the region of chromosome 7containing the human OB gene is depicted, as deduced by SEGMAP/Version3.29 (Green and Green, 1991a; C. L. Magness and P. Green, unpublisheddata). The 19 uniquely-ordered STSs (see Table 1) are listed along thetop. The 8 microsatellite-specific STSs are indicated with stars (seeTable 2). Also indicated are the STSs corresponding to the PAX4 and OBgenes as well as the predicted positions of the centromere (CEN) and 7qtelomere (TEL) relative to the contig. Each of the 43 YAC clones isdepicted by a horizontal bar, with its name given to the left andestimated YAC size (in kb, measured by pulsed-field gel electrophoresis)provided in parenthesis. The presence of an STS in a YAC is indicated bya darkened circle at the appropriate position. When an STS correspondsto the insert end of a YAC, a square is placed around the correspondingcircle, both along the top (near the STS name) and at the end of the YACfrom which it was derived. For the 5 YACs at the bottom (below thehorizontal dashed line), 1 or more STS(s) expected to be present (basedon the established STS order) was not detected [as assessed by testingthe individual YACs with the corresponding STS-specific PCR assay(s) atleast twice], and these are depicted as open circles at the appropriatepositions. Most of the YACs were isolated from a human-hamster hybridcell-derived library (Green et al. 1995a), with their original names asindicated. The remaining YACs were isolated from total human genomiclibraries, and their original library locations are provided in Table 3.Boxes are placed around the names of the 3 YACs (yWSS691, yWSS999, andyWSS2935) that were found by FISH analysis to map to 7q31.3. The contigis displayed in its uncomputed form, where YAC sizes are not used toestimate clone overlaps or STS spacing, and all of the STSs aretherefore spaced in an equidistant fashion. In the computed form, whereYAC sizes are used to estimate the relative distance separating eachpair of adjacent STSs as well as the extent of clone overlaps, the totalYAC contig appears to span just over 2 Mb.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984). Of particular relevance tothe present invention are strategies for isolating, cloning, sequencing,analyzing, and characterizing a gene or nucleic acid based on the wellknown polymerase chain reaction (PCR) techniques.

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The term “body weight modulator”, “modulator”, “modulators”, and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refers in oneinstance to both nucleotides and to proteinaceous material, the latterincluding both single or multiple proteins. More specifically, theaforementioned terms extend to the nucleotides and to the DNA having thesequences described herein and presented in FIG. 1 (SEQ ID NO:1), andFIG. 2 (SEQ ID NO:3). Likewise, the proteins having the amino acidsequence data described herein and presented in FIG. 1 (SEQ ID NO:2),and FIG. 3 (SEQ ID NO:4) are likewise contemplated, as are the profileof activities set forth with respect to all materials both herein and inthe claims. Accordingly, nucleotides displaying substantially equivalentor altered activity are likewise contemplated, including substantiallyhomologous analogs and allelic variations. Likewise, proteins displayingsubstantially equivalent or altered activity, including proteinsmodified deliberately, as for example, by site-directed mutagenesis, oraccidentally through mutations in hosts that produce the modulators arelikewise contemplated.

The terms “protein,” which refers to the naturally occurringpolypeptide, and “polypeptide” are used herein interchangeably withrespect to the ob gene product and variants thereof. The term “matureprotein” or “mature polypeptide” refers to the ob gene product with thesignal sequence (or a fusion protein partner) removed.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control.

A “vector” is a replicon, such as a plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

“Heterologous” DNA refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. Preferably, the heterologous DNAincludes a gene foreign to the cell.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. A cell has been “transformed”by exogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”) in either singlestranded form, or a double-stranded helix. Double stranded DNA-DNA,DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary or quaternary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide;or 30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; morepreferably at least about 15 nucleotides; most preferably the length isat least about 20 nucleotides.

“Homologous recombination” refers to the insertion of a foreign DNAsequence of a vector in a chromosome. Preferably, the vector targets aspecific chromosomal site for homologous recombination. For specifichomologous recombination, the vector will contain sufficiently longregions of homology to sequences of the chromosome to allowcomplementary binding and incorporation of the vector into thechromosome. Longer regions of homology, and greater degrees of sequencesimilarity, may increase the efficiency of homologous recombination.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNAsequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNAsequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be expressed on the surface of a cell. This sequenceencodes a signal peptide, N-terminal to the mature polypeptide, thatdirects the host cell to translocate the polypeptide. The term“translocation signal sequence” is also used herein to refer to thissort of signal sequence. Translocation signal sequences can be foundassociated with a variety of proteins native to eukaryotes andprokaryotes, and are often functional in both types of organisms.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5′) of andin reading frame with the gene.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash.

A molecule is “antigenic” when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567, as well asantigen binding portions of antibodies, including Fab, F(ab′), and Fr(including single chain antibodies). Accordingly, the phrase “antibodymolecule” in its various grammatical forms as used herein contemplatesboth an intact immunoglobulin molecule and an immunologically activeportion of an immunoglobulin molecule containing the antibody combiningsite. An “antibody combining site” is that structural portion of anantibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

A composition comprising “A” (where “A” is a single protein, DNAmolecule, vector, recombinant host cell, etc.) is substantially free of“B” (where “B” comprises one or more contaminating proteins, DNAmolecules, vectors, etc., but excluding racemic forms of A) when atleast about 75% by weight of the proteins, DNA, vectors (depending onthe category of species to which A and B belong) in the composition is“A”. Preferably, “A” comprises at least about 90% by weight of the A+Bspecies in the composition, most preferably at least about 99% byweight. It is also preferred that a composition, which is substantiallyfree of contamination, contain only a single molecular weight specieshaving the activity or characteristic of the species of interest.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15 percent, preferably byat least 50 percent, more preferably by at least 90 percent, and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in the host.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvants include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Preferably, the adjuvant is pharmaceutically acceptable.

In its primary aspect, the present invention is directed to theidentification of materials that function as modulators of mammalianbody weight. In particular, the invention concerns the isolation,purification and sequencing of certain nucleic acids that correspond tothe ob gene in both mice and humans, as well as the correspondingpolypeptides expressed by these nucleic acids. The invention thuscomprises the discovery of nucleic acids having the nucleotide sequencesset forth in FIG. 1 (SEQ ID NO:1) and FIG. 2 (SEQ ID NO:3), and todegenerate variants, alleles and fragments thereof, all possessing theactivity of modulating body weight and adiposity. The correspondence ofthe present nucleic acids to the ob gene portends their significantimpact on conditions such as obesity as well as other maladies anddysfunctions where abnormalities in body weight are a contributoryfactor. The invention extends to the proteins expressed by the nucleicacids of the invention, and particularly to those proteins set forth inFIG. 1 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4), FIG. 5 (SEQ ID NO:5), andFIG. 6 (SEQ ID NO:6), as well as conserved variants, active fragments,and cognate small molecules.

In particular, the present invention contemplates that naturallyoccurring fragments of the ob polypeptide may be important. The peptidesequence includes a number of sites that are frequently the target forproteolytic cleavage, e.g., arginine residues. It is possible that thefull length polypeptide may be cleaved at one or more such sites to formbiologically active fragments. Such biologically active fragments mayeither agonize or antagonize the functional activity of the obpolypeptide to reduce body weight.

As discussed earlier, the weight control modulator peptides or theirbinding partners or other ligands or agents exhibiting either mimicry orantagonism to them or control over their production, may be prepared inpharmaceutical compositions, with a suitable carrier and at a strengtheffective for administration by various means to a patient experiencingabnormal fluctuations in body weight or adiposity, either alone or aspart of an adverse medical condition such as cancer or AIDS, for thetreatment thereof. A variety of administrative techniques may beutilized, among them parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the recognition factors or their subunitsmay vary and in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the weight controlmodulators recognition factors and/or their subunits may possess certaindiagnostic applications and may for example, be utilized for the purposeof detecting and/or measuring conditions where abnormalities in bodyweight are or may be likely to develop. For example, the modulatorpeptides or their active fragments may be used to produce bothpolyclonal and monoclonal antibodies to themselves in a variety ofcellular media, by known techniques such as the hybridoma techniqueutilizing, for example, fused mouse spleen lymphocytes and myelomacells. These techniques are described in detail below. Likewise, smallmolecules that mimic or antagonize the activity(ies) of the receptorrecognition factors of the invention may be discovered or synthesized,and may be used in diagnostic and/or therapeutic protocols.

Panels of monoclonal antibodies produced against modulator peptides canbe screened for various properties; i.e., isotype, epitope, affinity,etc. Of particular interest are monoclonal antibodies that neutralizethe activity of the modulator peptides. Such monoclonals can be readilyidentified in activity assays for the weight modulators. High affinityantibodies are also useful when immunoaffinity purification of native orrecombinant modulator is possible.

Preferably, the anti-modulator antibody used in the diagnostic andtherapeutic methods of this invention is an affinity purified polyclonalantibody. More preferably, the antibody is a monoclonal antibody (mAb).In addition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions ofwhole antibody molecules.

As suggested earlier, a diagnostic method useful in the presentinvention comprises examining a cellular sample or medium by means of anassay including an effective amount of an antagonist to a modulatorprotein, such as an anti-modulator antibody, preferably anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable for the anti-modulator antibody moleculesused herein be in the form of Fab, Fab′, F(ab′), or F(v) portions orwhole antibody molecules. As previously discussed, patients capable ofbenefiting from this method include those suffering from cancer, AIDS,obesity or other condition where abnormal body weight is acharacteristic or factor. Methods for isolating the modulator andinducing anti-modulator antibodies and for determining and optimizingthe ability of anti-modulator antibodies to assist in the examination ofthe target cells are all well-known in the art.

The nucleic acids contemplated by the present invention extend asindicated, to other nucleic acids that code on expression for peptidessuch as those set forth in FIG. 1 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:4),FIG. 5 (SEQ ID NO:5), and FIG. 6 (SEQ ID NO:6) herein. Accordingly,while specific DNA has been isolated and sequenced in relation to the obgene, any animal cell potentially can serve as the nucleic acid sourcefor the molecular cloning of a gene encoding the peptides of theinvention. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA “library”), by chemical synthesis, bycDNA cloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (See, for example, Sambrook et al., 1989,supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U. K. Vol. I, II). Clones derived from genomic DNAmay contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will not contain intron sequences.Whatever the source, the gene should be molecularly cloned into asuitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, the genomic DNAcan be amplified using primers selected from the cDNA sequences.Alternatively, DNA fragments are generated, some of which will encodethe desired gene. The DNA may be cleaved at specific sites using variousrestriction enzymes. One may also use DNAse in the presence of manganeseto fragment the DNA, or the DNA can be physically sheared, as forexample, by sonication. The linear DNA fragments can then be separatedaccording to size by standard techniques, including but not limited to,agarose and polyacrylamide gel electrophoresis and columnchromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired ob or ob-like gene may be accomplishedin a number of ways. For example, if an amount of a portion of a ob orob-like gene or its specific RNA, or a fragment thereof, is availableand can be purified and labeled, the generated DNA fragments may bescreened by nucleic acid hybridization to the labeled probe (Benton andDavis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl.Acad. Sci. U.S.A. 72:3961). The present invention provides such nucleicacid probes, which can be conveniently prepared from the specificsequences disclosed herein, e.g., a hybridizable probe having anucleotide sequence corresponding to at least a 10, and preferably a 15,nucleotide fragment of the sequences depicted in FIG. 1 (SEQ ID NO:1) orFIG. 2 (SEQ ID NO:3). Preferably, a fragment is selected that is highlyunique to the modulator peptides of the invention. Those DNA fragmentswith substantial homology to the probe will hybridize. As noted above,the greater the degree of homology, the more stringent hybridizationconditions can be used. In one embodiment, low stringency hybridizationconditions are used to identify a homologous modulator peptide. However,in a preferred aspect, and as demonstrated experimentally herein, anucleic acid encoding a modulator peptide of the invention willhybridize to a nucleic acid having a nucleotide sequence such asdepicted in FIG. 1 (SEQ ID NO:1) or FIG. 2 (SEQ ID NO:3), or ahybridizable fragment thereof, under moderately stringent conditions;more preferably, it will hybridize under high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, tyrosine phosphatase activity orantigenic properties as known for the present modulator peptides. Forexample, the antibodies of the instant invention can conveniently beused to screen for homologs of modulator peptides from other sources.

A gene encoding a modulator peptide of the invention can also beidentified by mRNA selection, i.e., by nucleic acid hybridizationfollowed by in vitro translation. In this procedure, fragments are usedto isolate complementary mRNAs by hybridization. Such DNA fragments mayrepresent available, purified modulator DNA. Immunoprecipitationanalysis or functional assays (e.g., tyrosine phosphatase activity) ofthe in vitro translation products of the products of the isolated mRNAsidentifies the mRNA and, therefore, the complementary DNA fragments,that contain the desired sequences. In addition, specific mRNAs may beselected by adsorption of polysomes isolated from cells to immobilizedantibodies specifically directed against a modulator peptide.

A radiolabeled modulator peptide cDNA can be synthesized using theselected mRNA (from the adsorbed polysomes) as a template. Theradiolabeled mRNA or cDNA may then be used as a probe to identifyhomologous modulator peptide DNA fragments from among other genomic DNAfragments.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and Synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9, pUC or pUC plasmid derivatives, e.g., pGEXvectors, pET vectors, pmal-c, pFLAG, etc., and their derivatives,plasmids such as RP4; phage DNAs, e.g., the numerous derivatives ofphage λ, e.g., NM989, and other phage DNA, e.g., M13 and Filamentoussingle stranded phage DNA; yeast plasmids such as the 2μ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like. Ina preferred embodiment, expression of ob is achieved in methylotrophicyeast, e.g., Pichia pastoris yeast (see, e.g., International PatentPublication No. WO 90/03431, published 5 Apr. 1990, by Brierley et al.;International Patent Publication No. WO 90/10697, published 20 Sep.1990, by Siegel et al.). In a specific embodiment, infra, an expressionvector is engineered for expression of ob under control of the α-matingfactor signal sequence.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), theAOX 1 promoter of methylotrophic yeast, the promoters of the yeastα-mating factors, and other sequences known to control the expression ofgenes of prokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces; fungi such as yeasts(Saccharomyces, and methylotrophic yeast such as Pichia, Candida,Hansenula, and Torulopsis); and animal cells, such as CHO, R1.1, B-W andL-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1,BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plantcells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

In a specific embodiment, an ob fusion protein can be expressed. An obfusion protein comprises at least a functionally active portion of anon-ob protein joined via a peptide bond to at least a functionallyactive portion of an ob polypeptide. The non-ob sequences can be amino-or carboxy-terminal to the ob sequences. More preferably, for stableexpression of a proteolytically inactive ob fusion protein, the portionof the non-ob fusion protein is joined via a peptide bond to the aminoterminus of the ob protein. A recombinant DNA molecule encoding such afusion protein comprises a sequence encoding at least a functionallyactive portion of a non-ob protein joined in-frame to the ob codingsequence, and preferably encodes a cleavage site for a specificprotease, e.g., thrombin or Factor Xa, preferably at the ob-non-objuncture. In a specific embodiment, the fusion protein is expressed inEscherichia coli or in P. pastoris.

In a specific embodiment, infra, vectors were prepared to express themurine and human ob genes, with and without the codon for gln-49, inbacterial expression systems and yeast (Pichia) expression systems asfusion proteins. The ob gene is prepared with an endonuclease cleavagesite, e.g., using PCR and novel primers. It is desirable to confirmsequences generated by PCR, since the probability of including a pointmutation is greater with this technique. A plasmid containing ahistidine tag (HIS-TAG) and a proteolytic cleavage site is used. Thepresence of the histidine makes possible the selective isolation ofrecombinant proteins on a Ni-chelation column, or by affinitypurification. The proteolytic cleavage site, in a specific embodiment,infra, a thrombin cleavage site, is engineered so that treatment withthe protease, e.g., thrombin, will release the full length mature (i.e.,lacking a signal sequence) ob polypeptide.

In another aspect, the pGEX vector (Smith and Johnson, 1988, Gene67:31-40) can be used. This vector fuses the schistosoma japonicumglutathionine S-transferase cDNA to the sequence of interest. Bacterialproteins are harvested and recombinant proteins can be quickly purifiedon a reduced glutathione affinity column. The GST carrier cansubsequently be cleaved from fusion proteins by digestion withsite-specific proteases. After cleavage, the carrier and uncleavedfusion protein can be removed by absorption on glutathione agarose.Difficulty with the system occasionally arises when the encoded proteinis insoluble in aqueous solutions.

Expression of recombinant proteins in bacterial systems may result inincorrect folding of the expressed protein, requiring refolding. Therecombinant protein can be refolded prior to or after cleavage to form afunctionally active ob polypeptide. The ob polypeptide may be refoldedby the steps of (i) incubating the protein in a denaturing buffer thatcontains a reducing agent, and then (ii) incubating the protein in abuffer that contains an oxidizing agent, and preferably also contains aprotein stabilizing agent or a chaotropic agent, or both. Suitable redox(reducing/oxidizing) agent pairs include, but are not limited to,reduced glutathione/glutathione disulfide, cystine/cysteine,cystamine/cysteamine, and 2-mercaptoethanol/2-hydroxyethyldisulfide. Ina particular aspect, the fusion protein can be solubilized in adenaturant, such as urea, prior to exchange into the reducing buffer. Inpreferred embodiment, the protein is also purified, e.g., by ionexchange or Ni-chelation chromatography, prior to exchange into thereducing buffer. Denaturing agents include but are not limited to ureaand guanidine-HCl. The recombinant protein is then diluted about atleast 10-fold, more preferably about 100-fold, into an oxidizing bufferthat contains an oxidizing agent, such as but not limited to 0.1 MTris-HCl, pH 8.0, 1 mM EDTA, 0.15 M NaCl, 0.3 M oxidized glutathione.The fusion protein is then incubated for about 1 to about 24 hours,preferably about 2 to about 16 hours, at room temperature in theoxidizing buffer. The oxidizing buffer may comprise a proteinstabilizing agent, e.g., a sugar, an alcohol, or ammonium sulfate. Theoxidizing buffer may further comprises a chaotropic agent at lowconcentration, to destabilize incorrect intermolecular interactions andthus promote proper folding. Suitable chaotropic agents include but arenot limited to a detergent, a polyol, L-arginine, guanidine-HCl andpolyethylene glycol (PEG). It is important to use a low enoughconcentration of the chaotropic agent to avoid denaturing the protein.The refolded protein can be concentrated by at least about 10-fold, morepreferably by the amount it was diluted into the oxidizing buffer.

Bacterial fermentation processes can also result in a recombinantprotein preparation that contains unacceptable levels of endotoxins.Therefore, the invention contemplates removal of such endotoxins, e.g.,by using endotoxin-specific antibodies or other endotoxin bindingmolecules. The presence of endotoxins can be determined by standardtechniques, such as by employing E-TOXATE Reagents (Sigma), or withbioassays.

In addition to the specific example, the present inventors contemplateuse of baculovirus, mammalian, and yeast expression systems to expressthe ob protein. For example, in baculovirus expression systems, bothnon-fusion transfer vectors, such as but not limited to pVL941 (BamH1cloning site; Summers), pVL1393 (BamH1, SmaI, XbaI, EcoRI, NotI, XmaIII,BglII, and PstI cloning site; Invitrogen), pVL1392 (BglII, PstI, NotI,XmaIII, EcoRI, XbaI, SmaI, and BamH1 cloning site; Summers andInvitrogen), and pBlueBacIII (BamH1, BglII, PstI, NcoI, and HindIIIcloning site, with blue/white recombinant screening possible;Invitrogen), and fusion transfer vectors, such as but not limited topAc700 (BamH1 and KpnI cloning site, in which the BamH1 recognition sitebegins with the initiation codon; Summers), pAc701 and pAc702 (same aspAc700, with different reading frames), pAc360 (BamH1 cloning site 36base pairs downstream of a polyhedrin initiation codon;Invitrogen(195)), and pBlueBacHisA, B, C (three different readingframes, with BamH1, BglII, PstI, NcoI, and HindIII cloning site, anN-terminal peptide for ProBond purification, and blue/white recombinantscreening of plaques; Invitrogen (220)).

Mammalian expression vectors contemplated for use in the inventioninclude vectors with inducible promoters, such as dihydrofolatereductase (DHFR), e.g., any expression vector with a DHFR expressionvector, or a DHFR/methotrexate co-amplification vector, such as pED(PstI, SalI, SbaI, SmaI, and EcoRI cloning site, with the vectorexpressing both the cloned gene and DHFR; see Kaufman, Current Protocolsin Molecular Biology, 16.12, 1991). Alternatively, a glutaminesynthetase/methionine sulfoximine co-amplification vector, such as pEE14(HindIII, XbaI, SmaI, SmaI, EcoRI, and MI cloning site, in which thevector expresses glutamine synthase and the cloned gene; Celltech). Inanother embodiment, a vector that directs episomal expression undercontrol of Epstein Barr Virus (EBV) can be used, such as pREP4 (BamH1,SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII, and KpnI cloning site,constitutive RSV LTR promoter, hygromycin selectable marker;Invitrogen), pCEP4 (BamH1, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII,and KpnI cloning site, constitutive hCMV immediate early gene,hygromycin selectable marker; Invitrogen), pMEP4 (KpnI, PvuI, NheI,HindIII, NotI, XhoI, SfiI, BamH1 cloning site, induciblemethallothionein IIa gene promoter, hygromycin selectable marker:Invitrogen), pREP8 (BamH1, XhoI, NotI, HindIII, NheI, and KpnI cloningsite, RSV LTR promoter, histidinol selectable marker; Invitrogen), pREP9(KpnI, NheI, HindIII, NotI, XhoI, SfiI, and BamHI cloning site, RSV LTRpromoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV LTRpromoter, hygromycin selectable marker, N-terminal peptide purifiablevia ProBond resin and cleaved by enterokinase; Invitrogen). Selectablemammalian expression vectors for use in the invention include pRc/CMV(HindIII, BstXI, NotI, SbaI, and ApaI cloning site, G418 selection;Invitrogen), pRc/RSV (HindIII, SpeI, BstXI, Nod, XbaI cloning site, G418selection; Invitrogen), and others. Vaccinia virus mammalian expressionvectors (see, Kaufman, supra) for use according to the invention includebut are not limited to pSC11 (SmaI cloning site, TK- and β-galselection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI, NheI,SacII, KpnI, and HindIII cloning site; TK- and β-gal selection), andpTKgptF1S (EcoRI, PstI, SalI, AccI, HindIII, SbaI, BamHI, and HpAcloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention toexpress ob polypeptide. For example, the non-fusion pYES2 vector (XbaI,SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, KpnI, and HindIIIcloning sit; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, Shot,NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning site,N-terminal peptide purified with ProBond resin and cleaved withenterokinase; Invitrogen), to mention just two, can be employedaccording to the invention.

It is further intended that body weight modulator peptide analogs may beprepared from nucleotide sequences derived within the scope of thepresent invention. Analogs, such as fragments, may be produced, forexample, by pepsin digestion of weight modulator peptide material. Otheranalogs, such as muteins, can be produced by standard site-directedmutagenesis of weight modulator peptide coding sequences. Analogsexhibiting “weight modulator activity” such as small molecules, whetherfunctioning as promoters or inhibitors, may be identified by known invivo and/or in vitro assays.

In addition to recombinant expression of ob polypeptide, the presentinvention envisions and fully enables preparation of ob polypeptide, orfragments thereof, using the well known and highly developed techniquesof solid phase peptide synthesis. The invention contemplates using boththe popular Boc and Fmoc, as well as other protecting group strategies,for preparing ob polypeptide or fragments thereof. Various techniquesfor refolding and oxidizing the cysteine side chains to form a disulfidebond are also well known in the art.

Derivatives of Ob Peptides

Generally, the present protein (herein the term “protein” is used toinclude “peptide”, unless otherwise indicated) may be derivatized by theattachment of one or more chemical moieties to the protein moiety. Thechemically modified derivatives may be further formulated forintraarterial, intraperitoneal, intramuscular subcutaneous, intravenous,oral, nasal, pulmonary, topical or other routes of administration.Chemical modification of biologically active proteins has been found toprovide additional advantages under certain circumstances, such asincreasing the stability and circulation time of the therapeutic proteinand decreasing immunogenicity. See U.S. Pat. No. 4,179,337, Davis etal., issued Dec. 18, 1979. For a review, see Abuchowski et al., inEnzymes as Drugs. (J. S. Holcerberg and J. Roberts, eds. pp. 367-383(1981)). A review article describing protein modification and fusionproteins is Francis, Focus on Growth Factors 3: 4-10 (May 1992)(published by Mediscript, Mountview Court, Friern Barnet Lane, LondonN20, OLD, UK).

Chemical Moieties for Derivatization

The chemical moieties suitable for derivatization may be selected fromamong water soluble polymers. The polymer selected should be watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable. One skilled in the art willbe able to select the desired polymer based on such considerations aswhether the polymer/protein conjugate will be used therapeutically, andif so, the desired dosage, circulation time, resistance to proteolysis,and other considerations. For the present proteins and peptides, thesemay be ascertained using the assays provided herein.

Polymer Molecules

The water soluble polymer may be selected from the group consisting of,for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayadvantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Polymer/Protein Ratio

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

Attachment of the Chemical Moiety to the Protein

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art. E.g., EP 0 401 384 hereinincorporated by reference (coupling PEG to G-CSF), see also Malik etal., Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue: Sulfhydrl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecule(s). Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group. Attachment at residues important forreceptor binding should be avoided if receptor binding is desired.

N-Terminally Chemically Modified Proteins

One may specifically desire N-terminally chemically modified protein.Using polyethylene glycol as an illustration of the presentcompositions, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminalchemically modification may be accomplished by reductive alkylationwhich exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at pH which allows one to take advantage of the pK_(a)differences between the ε-amino groups of the lysine residues and thatof the α-amino group of the N-terminal residue of the protein. By suchselective derivatization attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolproprionaldehyde, containing a single reactive aldehyde, may be used.

As mentioned above, a DNA sequence encoding weight modulator peptides asdisclosed herein can be prepared synthetically rather than cloned. TheDNA sequence can be designed with the appropriate codons for the weightmodulator peptide amino acid sequences. In general, one will selectpreferred codons for the intended host if the sequence will be used forexpression. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature, 292:756 (1981);Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem.,259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express weight modulator analogs or “muteins”. Alternatively, DNAencoding muteins can be made by site-directed mutagenesis of nativemodulator genes or cDNAs, and muteins can be made directly usingconventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs ofthe ob polypeptide with unnatural amino acids.

The present invention extends to the preparation of antisensenucleotides and ribozymes that may be used to interfere with theexpression of the weight modulator proteins at the translational level.This approach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (See Weintraub, 1990;Marcus-Sekura, 1988). In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into weight modulator peptide-producing cells. Antisense methodshave been used to inhibit the expression of many genes in vitro(Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type (Hasselhoff and Gerlach, 1988). Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against and ribozymes that cleave mRNAs for weight modulatorproteins and their ligands, thus inhibiting expression of the ob gene,and leading to increased weight gain and adiposity.

The present invention also relates to a variety of diagnosticapplications, including methods for detecting the presence of conditionsand/or stimuli that impact abnormalities in body weight or adiposity, byreference to their ability to elicit the activities which are mediatedby the present weight modulators. As mentioned earlier, the weightmodulator peptides can be used to produce antibodies to themselves by avariety of known techniques, and such antibodies could then be isolatedand utilized as in tests for the presence of particular transcriptionalactivity in suspect target cells.

Antibody(ies) to the body weight modulators, i.e., the ob polypeptide,can be produced and isolated by standard methods including the wellknown hybridoma techniques. For convenience, the antibody(ies) to theweight modulators will be referred to herein as Ab₁ and antibody(ies)raised in another species as Ab₂.

According to the invention, ob polypeptide produced recombinantly or bychemical synthesis, and fragments or other derivatives or analogsthereof, including fusion proteins, may be used as an immunogen togenerate antibodies that recognize the ob polypeptide. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to ob polypeptide a recombinant PTP or derivativeor analog thereof. For the production of antibody, various host animalscan be immunized by injection with the ob polypeptide, or a derivative(e.g., fragment or fusion protein) thereof, including but not limited torabbits, mice, rats, sheep, goats, etc. In one embodiment, the obpolypeptide or fragment thereof can be conjugated to an immunogeniccarrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin(KLH). Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the obpolypeptide, or fragment, analog, or derivative thereof, any techniquethat provides for the production of antibody molecules by continuouscell lines in culture may be used. These include but are not limited tothe hybridoma technique originally developed by Kohler and Milstein(1975, Nature 256:495-497), as well as the trioma technique, the humanB-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),and the EBV-hybridoma technique to produce human monoclonal antibodies(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for an ob polypeptide together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in therapy ofhuman diseases or disorders (described infra), since the human orhumanized antibodies are much less likely than xenogenic antibodies toinduce an immune response, in particular an allergic response,themselves.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce ob polypeptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for an ob polypeptide, or itsderivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an ob polypeptide, one may assay generatedhybridomas for, a product which binds to an ob polypeptide fragmentcontaining such epitope. For selection of an antibody specific to an obpolypeptide from a particular species of animal, one can select on thebasis of positive binding with ob polypeptide expressed by or isolatedfrom cells of that species of animal.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the ob polypeptide, e.g.,for Western blotting, imaging ob polypeptide in situ, measuring levelsthereof in appropriate physiological samples, etc.

In a specific embodiment, antibodies that agonize or antagonize theactivity of ob polypeptide can be generated. Such antibodies can betested using the assays described infra for identifying ligands.

Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

In a specific embodiment, antibodies are developed by immunizing rabbitswith synthetic peptides predicted by the protein sequence or withrecombinant proteins made using bacterial expression vectors. The choiceof synthetic peptides is made after careful analysis of the predictedprotein structure, as described above. In particular, peptide sequencesbetween putative cleavage sites are chosen. Synthetic peptides areconjugated to a carrier such as KLH hemocyanin or BSA using carbodiimideand used in Freunds adjuvant to immunize rabbits. In order to preparerecombinant protein, the gex vector can be used to express thepolypeptide (Smith and Johnson, supra). Alternatively, one can use onlyhydrophilic domains to generate the fusion protein. The expressedprotein will be prepared in quantity and used to immunize rabbits inFreunds adjuvant.

The presence of weight modulator in cells can be ascertained by theusual immunological procedures applicable to such determinations. Anumber of useful procedures are known. Three such procedures which areespecially useful utilize either the receptor recognition factor labeledwith a detectable label, antibody Ab₁ labeled with a detectable label,or antibody Ab₂ labeled with a detectable label. The procedures may besummarized by the following equations wherein the asterisk indicatesthat the particle is labeled, and “WM” stands for the weight modulator:WM*+Ab ₁ =WM*Ab ₁  A.WM+Ab*=WMAb ₁  B.WM+Ab ₁ +Ab ₂ *=Ab ₁ WMAb ₂*  C.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, Procedure A, isdescribed in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure B isrepresentative of the well known competitive assay techniques. ProcedureC, the “sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006and 4,016,043. Still other procedures are known such as the “doubleantibody”, or “DASP” procedure.

In each instance, the weight modulators form complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab₂ isthat it will react with Ab₁. This is because Ab₁ raised in one mammalianspecies has been used in another species as an antigen to raise theantibody Ab₂. For example, Ab₂ may be raised in goats using rabbitantibodies as antigens. Ab₂ therefore would be anti-rabbit antibodyraised in goats. For purposes of this description and claims, Ab₁ willbe referred to as a primary or anti-weight modulator antibody, and Ab₂will be referred to as a secondary or anti-Ab₁ antibody.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others.

A number of fluorescent materials are known and can be utilized aslabels. These include, for example, fluorescein, rhodamine and auramine.A particular detecting material is anti-rabbit antibody prepared ingoats and conjugated with fluorescein through an isothiocyanate.

The weight modulators or their binding partners can also be labeled witha radioactive element or with an enzyme. The radioactive label can bedetected by any of the currently available counting procedures. Thepreferred isotope may be selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system that is to be utilized in accordance with thepresent invention, is known as a receptor assay. In a receptor assay,the material to be assayed is appropriately labeled and then certaincellular test colonies are inoculated with a quantity of both thelabeled and unlabeled material after which binding studies are conductedto determine the extent to which the labeled material binds to the cellreceptors. In this way, differences in affinity between materials can beascertained.

Accordingly, a purified quantity of the weight modulator may beradiolabeled and combined, for example, with antibodies or otherinhibitors thereto, after which binding studies would be carried out.Solutions would then be prepared that contain various quantities oflabeled and unlabeled uncombined weight modulator, and cell sampleswould then be inoculated and thereafter incubated. The resulting cellmonolayers are then washed, solubilized and then counted in a gammacounter for a length of time sufficient to yield a standard error of<5%. These data are then subjected to Scatchard analysis after whichobservations and conclusions regarding material activity can be drawn.While the foregoing is exemplary, it illustrates the manner in which areceptor assay may be performed and utilized, in the instance where thecellular binding ability of the assayed material may serve as adistinguishing characteristic. In turn, a receptor assay will beparticularly useful in the identification of the specific receptors tothe present modulators, such as the db receptor.

A further assay useful and contemplated in accordance with the presentinvention is known as a “cis/trans” assay. Briefly, this assay employstwo genetic constructs, one of which is typically a plasmid thatcontinually expresses a particular receptor of interest when transfectedinto an appropriate cell line, and the second of which is a plasmid thatexpresses a reporter such as luciferase, under the control of areceptor/ligand complex. Thus, for example, if it is desired to evaluatea compound as a ligand for a particular receptor, one of the plasmidswould be a construct that results in expression of the receptor in thechosen cell line, while the second plasmid would possess a promoterlinked to the luciferase gene in which the response element to theparticular receptor is inserted. If the compound under test is anagonist for the receptor, the ligand will complex with the receptor, andthe resulting complex will bind the response element and initiatetranscription of the luciferase gene. The resulting chemiluminescence isthen measured photometrically, and dose response curves are obtained andcompared to those of known ligands. The foregoing protocol is describedin detail in U.S. Pat. No. 4,981,784 and PCT International PublicationNo. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of predetermined transcriptional activity orpredetermined transcriptional activity capability in suspected targetcells. In accordance with the testing techniques discussed above, oneclass of such kits will contain at least the labeled weight modulator orits binding partner, for instance an antibody specific thereto, anddirections, of course, depending upon the method selected, e.g.,“competitive”, “sandwich”, “DASP” and the like. The kits may alsocontain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for predetermined transcriptionalactivity, comprising:

-   -   (a) a predetermined amount of at least one labeled        immunochemically reactive component obtained by the direct or        indirect attachment of the present weight modulator or a        specific binding partner thereto, to a detectable label;    -   (b) other reagents; and    -   (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

-   -   (a) a known amount of the weight modulator as described above        (or a binding partner) generally bound to a solid phase to form        an immunosorbent, or in the alternative, bound to a suitable        tag, or plural such end products, etc. (or their binding        partners) one of each;    -   (b) if necessary, other reagents; and    -   (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive”, “sandwich”, “double antibody”, etc.), andcomprises:

-   -   (a) a labeled component which has been obtained by coupling the        weight modulator to a detectable label;    -   (b) one or more additional immunochemical reagents of which at        least one reagent is a ligand or an immobilized ligand, which        ligand is selected from the group consisting of:        -   (i) a ligand capable of binding with the labeled component            (a);        -   (ii) a ligand capable of binding with a binding partner of            the labeled component (a);        -   (iii) a ligand capable of binding with at least one of the            component(s) to be determined; and        -   (iv) a ligand capable of binding with at least one of the            binding partners of at least one of the component(s) to be            determined; and    -   (c) directions for the performance of a protocol for the        detection and/or determination of one or more components of an        immunochemical reaction between the weight modulator and a        specific binding partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to mimic or antagonize the activity of the weightmodulator may be prepared. The weight modulator may be introduced into atest system, and the prospective drug may also be introduced into theresulting cell culture, and the culture thereafter examined to observeany changes in the activity of the cells, due either to the addition ofthe prospective drug alone, or due to the effect of added quantities ofthe known weight modulator.

As stated earlier, the molecular cloning of the ob gene described hereinhas led to the identification of a class of materials that function onthe molecular level to modulate mammalian body weight. The discovery ofthe modulators of the invention has important implications for thediagnosis and treatment of nutritional disorders including, but notlimited to, obesity, weight loss associated with cancer and thetreatment of diseases associated with obesity such as hypertension,heart disease and Type II diabetes. In addition, there are potentialagricultural uses for the gene product in cases where one might wish tomodulate the body weight of domestic animals. Finally, to the extentthat one or more of the modulators of the invention are secretedmolecules, they can be used biochemically to isolate their receptorusing the technology of expression cloning. The discussion that followswith specific reference to the ob gene bears general applicability tothe class of modulators that a part of the present invention, and istherefore to be accorded such latitude and scope of interpretation.

Therapeutic Implications

In the simplest analysis the ob gene determines body weight in mammals,in particular mice and man. The ob gene and, correspondingly, cognatemolecules, appear to be part of a signaling pathway by which adiposetissue communicates with the brain and the other organs. It is believedthat the ob polypeptide is itself a signaling molecule, i.e., a hormone.Alternatively ob may be responsible for the generation of a metabolicsignal, e.g., a stimulating hormone or an enzyme that catalyzesactivation or synthesis of a peptide or steroid hormone. The mostimportant piece of information for distinguishing between thesepossibilities or considering alternative hypothesis, is the complete DNAsequence of the RNA and its predicted protein sequence. Irrespective ofits biochemical function the genetic data suggest that increasedactivity of ob would result in weight loss while decreased activitywould be associated with weight gain. The means by which the activity ofob can be modified so as to lead to a therapeutic effect depends on itsbiochemical function.

Administration of recombinant ob polypeptide can result in weight loss.Recombinant protein can be prepared using standard bacterial and/ormammalian expression vectors, all as stated in detail earlier herein.Reduction of ob polypeptide activity (by developing antagonists,inhibitors, or antisense molecules) should result in weight gain asmight be desirable for the treatment of the weight loss associated withcancer, AIDS or anorexia nervosa. Modulation of ob activity can beuseful for reducing body weight (by increasing its activity) orincreasing body weight (by decreasing its activity).

The ob polypeptide, or functionally active fragment thereof, or anantagonist thereof, can be administered orally or parenterally,preferably parenterally. Because metabolic homeostasis is a continuousprocess, controlled release administration of ob polypeptide ispreferred. For example, the polypeptide may be administered usingintravenous infusion, an implantable osmotic pump, a transdermal patch,liposomes, or other modes of administration. In one embodiment, a pumpmay be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.Engl. J. Med. 321:574 (1989)). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol.Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard etal., J. Neurosurg. 71:105 (1989)). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, i.e., the brain, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533 (1990)). Inanother embodiment, the therapeutic compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In a further aspect, recombinant cells that have been transformed withthe ob gene and that express high levels of the polypeptide can betransplanted in a subject in need of ob polypeptide. Preferablyautologous cells transformed with ob are transplanted to avoidrejection; alternatively, technology is available to shieldnon-autologous cells that produce soluble factors within a polymermatrix that prevents immune recognition and rejection.

Thus, the ob polypeptide can be delivered by intravenous, intraarterial,intraperitoneal, intramuscular, or subcutaneous routes ofadministration. Alternatively, the ob polypeptide, properly formulated,can be administered by nasal or oral administration. A constant supplyof ob can be ensured by providing a therapeutically effective dose(i.e., a dose effective to induce metabolic changes in a subject) at thenecessary intervals, e.g., daily, every 12 hours, etc. These parameterswill depend on the severity of the disease condition being treated,other actions, such as diet modification, that are implemented, theweight, age, and sex of the subject, and other criteria, which can bereadily determined according to standard good medical practice by thoseof skill in the art.

Alternatively, the ob gene could be introduced into human fat cells todevelop gene therapy for obesity. Such therapy would be expected todecrease body weight. Conversely, introduction of antisense constructsinto human fat cells would reduce the levels of active ob polypeptideand would be predicted to increase body adiposity.

If ob is an enzyme, strategies have begun to be developed for theidentification of the substrate and product of the catalyzed reactionthat would make use of the recombinant protein. The rationale for thisstrategy is as follows: If ob is an enzyme that catalyzes a particularreaction in adipose tissue, then fat cells from ob mice should have highlevels of the substrate and very little product. Since it ishypothesized that db mice are resistant to the product of this reaction,fat cells from db mice should have high levels of the reaction product.Thus, comparisons of lipid and peptide extracts of ob and db adiposetissue using gas chromatography or other chromatographic methods shouldallow the identification of the product and substrate of the keychemical reaction. The prediction would be that the recombinant obprotein would catalyze this reaction. The product of this reaction wouldthen be a candidate for a signaling molecule that modulates body weight.

As noted above, the functional activity of the ob polypeptide can beeffected transgenically, e.g., by gene therapy. In this respect, atransgenic mouse model can be used. The ob gene can be used incomplementation studies employing transgenic mice. Transgenic vectors,including viral vectors, or cosmid clones (or phage clones)corresponding to the wild type locus of candidate gene, can beconstructed using the isolated ob gene. Cosmids may be introduced intotransgenic mice using published procedures (Jaenisch, Science 240,1468-1474, 1988). The constructs are introduced into fertilized eggsderived from an intercross between F1 progeny of a C57BL/6J ob/ob X DBAintercross. These crosses require the use of C57BL/6J ob/ob ovariantransplants to generate the F1 animals. DBA/2J mice are used as thecounterstrain because they have a nonagouti coat color which isimportant when using the ovarian transplants. Genotype at the ob loci incosmid transgenic animals can be determined by typing animals withtightly linked RFLPs or microsatellites which flank the mutation andwhich are polymorphic between the progenitor strains. Complementationwill be demonstrated when a particular construct renders a geneticallyobese F2 animal (as scored by RFLP analysis) lean and nondiabetic. Underthese circumstances, final proof of complementation will require thatthe ob/ob or db/db animal carrying the transgene be mated to the ob/obor db/db ovarian transplants. In this cross, all N2 animals which do notcarry the transgene will be obese and insulin resistant/diabetic, whilethose that do carry the transgene will be lean and have normal glucoseand insulin concentrations in plasma. In a genetic sense, the transgeneacts as a suppressor mutation.

Alternatively, ob genes can be tested by examining their phenotypiceffects when express in antisense orientation in wild-type animals. Inthis approach, expression of the wild type allele is suppressed, whichleads to a mutant phenotype. RNA•RNAduplex formation (antisense•sense)prevents normal handling of mRNA, resulting in partial or completeelimination of wild-type gene effect. This technique has been used toinhibit Tk synthesis in tissue culture and to produce phenotypes of theKruppel mutation in Drosophila, and the shiverer mutation in mice (Izantand Weintraub, Cell 36, 1007-1015, 1984; Green et al., Annu. Rev.Biochem. 55, 569-597, 1986; Katsuki et al., Science 241, 593-595, 1988).An important advantage of this approach is that only a small portion ofthe gene need be expressed for effective inhibition of expression of theentire cognate mRNA. The antisense transgene will be placed undercontrol of its own promoter or another promoter expressed in the correctcell type, and placed upstream of the SV40 poly A site. This transgenewill be used to make transgenic mice. Transgenic mice will also be matedovarian transplants to test whether ob heterozygotes are more sensitiveto the effects of the antisense construct.

In the long term, the elucidation of the biochemical function of the obgene product (the ob polypeptide or protein) is useful for identifyingsmall molecule agonists and antagonists that affect its activity.

Pharmaceutical Compositions

In yet another aspect of the present invention, provided arepharmaceutical compositions of the above. Such pharmaceuticalcompositions may be for administration for injection, or for oral,pulmonary, nasal or other forms of administration. In general,comprehended by the invention are pharmaceutical compositions comprisingeffective amounts of protein or derivative products of the inventiontogether with pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hylauronic acid may also be used. Such compositionsmay influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the present proteins and derivatives.See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (E.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the protein (orchemically modified protein), and inert ingredients which allow forprotection against the stomach environment, and release of thebiologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized proteins. Protein may be chemically modified so that oraldelivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe protein (or peptide) molecule itself, where said moiety permits (a)inhibition of proteolysis; and (b) uptake into the blood stream from thestomach or intestine. Also desired is the increase in overall stabilityof the protein and increase in circulation time in the body. Examples ofsuch moieties include: Polyethylene glycol, copolymers of ethyleneglycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis,Soluble Polymer-Enzyme Adducts. In: “Enzymes as Drugs”, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., (1981), pp 367-383;Newmark, et al., J. Appl. Biochem. 4: 185-189 (1982). Other polymersthat could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.Preferred for pharmaceutical usage, as indicated above, are polyethyleneglycol moieties.

For the protein (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunem, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Controlled release formulation may be desirable. The drug could beincorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms i.e. gums. Slowly degenerating matricesmay also be incorporated into the formulation. Another form of acontrolled release of this therapeutic is by a method based on the Orostherapeutic system (Alza Corp.), i.e. the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entriccoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., PHARMACEUTICAL RESEARCH, VOL. 7, No. 6, pp. 565-569(1990); Adjei et al., International Journal of Pharmaceutics, Vol. 63,pp. 135-144 (1990)(leuprolide acetate); Braquet et al., Journal ofCardiovascular Pharmacology, Vol. 13, suppl. 5, s. 143-146(1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine, Vol.III, No. 3, pp. 206-212 (1989)(α1-antitrypsin); Smith et al., J. Clin.Invest., Vol. 84, pp. 1145-1146 (1989)(α1-proteinase); Oswein et al.,“Aerosolization of Proteins”, Proceedings of Symposium on RespiratoryDrug Delivery II, Keystone, Colo., March, 1990 (recombinant human growthhormone); Debs et al., The Journal of Immunology, Vol. 140, pp.3482-3488 (1988)(interferon-γ and tumor necrosis factor alpha) and Platzet al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent nebulizer, manufacturedby Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, North Carolina; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of protein (or derivative). Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy. Also, the use of liposomes,microcapsules or microspheres, inclusion complexes, or other types ofcarriers is contemplated. Chemically modified protein may also beprepared in different formulations depending on the type of chemicalmodification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise protein (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the protein (or derivative)suspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing protein (or derivative) and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The protein (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 μm (or microns), mostpreferably 0.5 to 5 μm, for most effective delivery to the distal lung.

Nasal Delivery

Nasal delivery of the protein (or derivative) is also contemplated.Nasal delivery allows the passage of the protein to the blood streamdirectly after administering the therapeutic product to the nose,without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

Methods of Treatment, Methods of Preparing a Medicament

In yet another aspect of the present invention, methods of treatment andmanufacture of a medicament are provided. Conditions alleviated ormodulated by the administration of the present derivatives are thoseindicated above.

Dosages

For all of the above molecules, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, will be able to ascertain proper dosing.Generally, for injection or infusion, dosage will be between 0.01 μg ofbiologically active protein/kg body weight, (calculating the mass of theprotein alone, without chemical modification), and 100 μg/kg (based onthe same). The dosing schedule may vary, depending on the circulationhalf-life of the protein or derivative used, and the formulation used.

Administration with Other Compounds

For therapy associated with obesity, one may administer the presentprotein (or derivatives) in conjunction with one or more pharmaceuticalcompositions used for treating other clinical complications of obesity,such as those used for treatment of diabetes (e.g., insulin), high bloodpressure, high cholesterol, and other adverse conditions incident toobesity. Also, other appetite suppressants may be co-administered, e.g.,amphetamines. Administration may be simultaneous (for example,administration of a mixture of the present protein and insulin) or maybe in serriatim.

Diagnostic Implications

The human cDNA clones that have recently been isolated have beensequenced as presented herein. This facilitates the determination of thecomplete sequence of the human gene (see FIG. 20). DNA sequences fromthe introns of the human ob gene have been obtained (FIG. 20), and thesehave been used to prepare PCR primers to PCR amplify the coding sequenceof the ob gene from human genomic DNA so as to identify mutations orallelic variants of the ob gene, all in accordance with protocolsdescribed in detail earlier herein. Specific PCR primers for amplifyinghuman genomic ob are described in a specific Example, infra.

The current hypothesis is that heterozygous mutations in the ob genewill be associated with mild/moderate obesity while homozygous mutationswould be associated with several DNA sequence based diagnostic testsobesity. If this is true, it would allow the ascertainment of people atrisk for the development of obesity and make possible the application ofdrug treatment and/or lifestyle changes before an increased body weightis fully developed.

Alternatively, the presence of microsatellites that segregate withmutant forms of human ob can be used for diagnosis. Various PCR primers,including those based on the nucleotide sequence provided in FIG. 20A,can be used in this respect.

The ob gene may also be useful diagnostically for measurements of itsencoded RNA and protein in nutritional disorders. It will be ofimportance to know, in a particular nutritional disorder, whether ob RNAand/or protein is unregulated or downregulated. Thus, if an obese personhas increased levels of ob, it would appear that the problem isdownstream of ob, while if ob is reduced, it would appear thatinappropriately low levels of ob may be cause of obesity (whether or notthe defect is in the ob gene). Conversely, if a cancer or AIDS patientwho lost weight had elevated levels of ob, it may be concluded thatinappropriately high expression of ob is responsible for the weightloss.

The cloned human cDNA will be of use for the measurement of the levelsof human ob RNA. In addition, recombinant human protein will be preparedand used to develop immunoassays to enable measurement of the fat andperhaps plasma levels of the ob protein.

Agricultural Applications

The ob gene can also be isolated from domestic animals, and thecorresponding ob polypeptide obtained thereby. In a specific example,infra, the a probe derived from the murine ob gene hybridizes tocorresponding homologous coding sequences from a large number of speciesof animals. As discussed for human therapies, recombinant proteins canalso be prepared and administered to domestic animals. Administration ofthe polypeptide can be implemented to produce leaner food animals, suchas beef cattle, swine, poultry, sheep, etc. Preferably, an autologous obpolypeptide is administered, although the invention contemplatesadministration of anti-autologous polypeptide as well. Since the obpolypeptide consists of approximately 160 amino acid residues, it maynot be highly immunogenic. Thus, administration of non-autologouspolypeptide may not result in an immune response.

Alternatively, the introduction of the cloned genes into transgenicdomestic animals would allow one to potentially decrease body weight andadiposity by overexpressing an ob transgene. The simplest means ofachieving this would be to target an ob transgene to fat using its ownor another fat specific promoter.

Conversely, increases in body fat might be desirable in othercircumstances such as for the development of Kobe beef or fatty liver tomake foie gras. This could be accomplished by targeting an antisense obtransgene to fat, or by using gene knockout technology. Alternatively,where an increase in body weight at percentage of fat is desired, aninhibitor or antagonist of the ob polypeptide can be administered. Suchinhibitors or antagonists include, but are not limited to, antibodiesreactive with the polypeptide, and fragments of the polypeptide thatbind but do not activate the ob receptor, i.e., antagonists of obpolypeptide.

The ob Receptor

Development of small molecule agonists and antagonists of the ob factorwill be greatly facilitated by the isolation of its receptor. This canbe accomplished by preparing active ob polypeptide and using it toscreen an expression library using standard methodology. Receptorbinding in the expression library can be tested by administeringrecombinant polypeptide prepared using either bacterial or mammalianexpression vectors, and observing the effects of short term andcontinuous administration of the recombinant polypeptide on the cells ofthe expression library, or by directly detecting binding of obpolypeptide to the cells.

As it is presently believed that the ob receptor is likely to be locatedin the hypothalamus and perhaps liver, preferably cDNA libraries fromthese tissues will be constructed in standard expression cloningvectors. These cDNA clones would next be introduced into COS cells aspools and the resulting transformants would be screened with activeligand to identify COS cells expressing the ob receptor. Positive clonescan then be isolated so as to recover the cloned receptor. The clonedreceptor would be used in conjunction with the ob ligand (assuming it isa hormone) to develop the necessary components for screening of smallmolecule modulators of ob.

Once a recombinant which expresses the ob receptor gene sequence isidentified, the recombinant ob receptor can be analyzed. This isachieved by assays based on the physical or functional properties of theob receptor, including radioactive labelling of the receptor followed byanalysis by gel electrophoresis, immunoassay, ligand binding, etc.Furthermore, antibodies to the ob receptor could be generated asdescribed above.

The structure of the ob receptor can be analyzed by various methodsknown in the art. Preferably, the structure of the various domains,particularly the ob binding site, is analyzed. Structural analysis canbe performed by identifying sequence similarity with other knownproteins, particular hormone and protein receptors. The degree ofsimilarity (or homology) can provide a basis for predicting structureand function of the ob receptor, or a domain thereof. In a specificembodiment, sequence comparisons can be performed with sequences foundin GenBank, using, for example, the FASTA and FASTP programs (Pearsonand Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-48).

The protein sequence can be further characterized by a hydrophilicityanalysis (e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A.78:3824). A hydrophilicity profile can be used to identify thehydrophobic and hydrophilic regions of the ob receptor protein, whichmay in turn indicate extracytoplasmic, membrane binding, andintracytoplasmic regions.

Secondary structural analysis (e.g., Chou and Fasman, 1974, Biochemistry13:222) can also be done, to identify regions of the ob receptor thatassume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as wellas open reading frame prediction and plotting, can also be accomplishedusing computer software programs available in the art.

By providing an abundant source of recombinant ob polypeptide, and theopportunity to isolate the ob receptor (i.e., the db gene product), thepresent invention enables quantitative structural determination of theactive conformation of the ob polypeptide and the ob receptor, ordomains thereof. In particular, enough material is provided for nuclearmagnetic resonance (NMR), infrared (IR), Raman, and ultraviolet (UV),especially circular dichroism (CD), spectroscopic analysis. Inparticular NMR provides very powerful structural analysis of moleculesin solution, which more closely approximates their native environment(Marion et al., 1983, Biochem. Biophys. Res. Comm. 113:967-974; Bar etal., 1985, J. Magn. Reson. 65:355-360; Kimura et al., 1980, Proc. Natl.Acad. Sci. U.S.A. 77:1681-1685). Other methods of structural analysiscan also be employed. These include but are not limited to X-raycrystallography (Engstom, A., 1974, Biochem. Exp. Biol. 11:7-13).

More preferably, co-crystals of ob polypeptide and ob receptor can bestudied. Analysis of co-crystals provides detailed information aboutbinding, which in turn allows for rational design of ligand agonists andantagonists. Computer modeling can also be used, especially inconnection with NMR or X-ray methods (Fletterick, R. and Zoller, M.(eds.), 1986, Computer Graphics and Molecular Modeling, in CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.).

Identification and isolation of a gene encoding an ob receptor of theinvention provides for expression of the receptor in quantities greaterthan can be isolated from natural sources, or in indicator cells thatare specially engineered to indicate the activity of a receptorexpressed after transfection or transformation of the cells. According,in addition to rational design of agonists and antagonists based on thestructure of ob polypeptide, the present invention contemplates analternative method for identifying specific ligands of ob receptor usingvarious screening assays known in the art.

Any screening technique known in the art can be used to screen for obreceptor agonists or antagonists. The present invention contemplatesscreens for small molecule ligands or ligand analogs and mimics, as wellas screens for natural ligands that bind to and agonize or antagonizeactivates ob receptor in vivo.

Knowledge of the primary sequence of the receptor, and the similarity ofthat sequence with proteins of known function, can provide an initialclue as the inhibitors or antagonists of the protein. Identification andscreening of antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” (Scott and Smith, 1990, Science249:386-390; Cwirla, et al., 1990, Proc. Natl. Acad. Sci., 87:6378-6382;Devlin et al., 1990, Science, 249:404-406), very large libraries can beconstructed (10⁶-10⁸ chemical entities). A second approach usesprimarily chemical methods, of which the Geysen method (Geysen et al.,1986, Molecular Immunology 23:709-715; Geysen et al. 1987, J.Immunologic Method 102:259-274) and the recent method of Fodor et al.(1991, Science 251, 767-773) are examples. Furka et al. (1988, 14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013;Furka, 1991, Int. J. Peptide Protein Res. 37:487-493), Houghton (U.S.Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat.No. 5,010,175, issued Apr. 23, 1991) describe methods to produce amixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries (Needels et al., 1993,“Generation and screening of an oligonucleotide encoded syntheticpeptide library,” Proc. Natl. Acad. Sci. USA 90:10700-4; Lam et al.,International Patent Publication No. WO 92/00252, each of which isincorporated herein by reference in its entirety), and the like can beused to screen for ob receptor ligands according to the presentinvention. With such libraries, receptor antagonists can be detectedusing cell that express the receptor without actually cloning the obreceptor (Lam et al., supra).

Alternatively, assays for binding of soluble ligand to cells thatexpress recombinant forms of the ob receptor ligand binding domain canbe performed. The soluble ligands can be provided readily as recombinantor synthetic ob polypeptide.

The screening can be performed with recombinant cells that express theob receptor, or alternatively, using purified receptor protein, e.g.,produced recombinantly, as described above. For example, the ability oflabeled, soluble or solubilized ob receptor that includes theligand-binding portion of the molecule, to bind ligand can be used toscreen libraries, as described in the foregoing references.

Example Section

The following outlines the method used to identify the genetic materialthat is exemplary of the present invention. This endeavor comprises foursequential steps: A) Genetic Mapping, B) Physical Mapping, C) CandidateGene Isolation, and D) Mutation detection. Following confirmation thatthe murine gene in object was isolated (Step D), the homologous humangene was sought, and both the murine and human genes and putativeproteins were characterized. The steps are summarized in greater detail,below.

A. Genetic Mapping

The ob mutation was segregated in genetic crosses and standard linkageanalysis was used to position the mutation relative to RFLPs(restriction fragment length polymorphisms). These data placed the obgene in an ˜5 cM interval on proximal mouse chromosome 6. (5 cM is ameasurement of genetic distance corresponding to 5 apparent geneticcrossovers per 100 animals.) A total of 771 informative meioses weregenerated and used in subsequent genetic mapping (Friedman et al.Genomics 11: 1054-1062, 1991). The genetic loci that were mappedrelative to ob were all previously published. The two closest RFLPsdescribed were defined by probes derived from the carboxypeptidase andmet oncogene genes.

The genetic resolution of the experiments described above was inadequateto clone ob, principally because none of the genetic markers were intight linkage. In order to identify the requisite tightly linked RFLPs,additional probes were isolated and the genetic cross was expanded. Amethod known as chromosome microdissection was used to isolate randompieces of DNA from proximal mouse chromosome 6 (Bahary et al., MammalianGenome 4: 511-515, 1993). Individual cloned probes were tested for tightlinkage to ob. On the basis of these studies one probe, D6Rck13, alsotermed psd3, was selected for further analysis owing to its geneticproximity to ob.

This probe was used to genotype 835 ob progeny from interspecific andintersubspecific crosses, which indicated that D6Rck13 is nonrecombinantin all 835 animals as reported in Bahary et al. In the course ofphysical mapping, a new polymorphic marker was identified from a cosmidsubclone derived from YAC 53A6. This new marker was positioned betweenD6Rck13 and the ob gene and was used to genotype the additional 771informative meioses from intraspecific intercross and backcross. Asingle animal #167 was identified to bear a recombination crossoverbetween ob and D6Rck39. These studies indicated that D6Rck39/D6RcK13 is˜0.06 cM from ob. An additional probe, Pax-4, was identified that was0.12 cM proximal to ob. Pax-4 was recombinant in two animals; #111 and420. Pax-4 is a pseudogene that was previously mapped to proximal mousechromosome 6 by Gruss and co-workers (Gruss et al. Genomics 11:424-434,1991). On this basis, it was determined that the ob gene resides in the˜0.2 cM interval between Pax-4 and D6Rck13. This led to efforts to clonethe interposing DNA in an effort to isolate ob.

B. Physical Mapping

The cloning of the DNA in this interval made use of yeast artificialchromosomes (YACs), a relatively new cloning vector that allows thecloning of long stretches of contiguous DNA often more than 1 millionbase pairs in length.

Firstly, yeast artificial chromosomes were isolated using D6Rck13 andPax-4. This was accomplished by preparing purified DNA probes and usingthem to isolate the corresponding YACs. These YACs (#8, 16, 107 and 24)were isolated and initially characterized, and on the basis of theresulting analyses it was concluded that YAC 16 was the YAC thatextended furthest distally, i.e., closest to ob. The key end of YAC #16was then recovered, and it was determined that this end was closer to obthan Pax-4. This end was termed 16M(+). This conclusion was reachedbecause it was shown that this probe was not recombinant in animal #420(as was Pax-4). This end was sequenced and used to develop a PCR assay.This PCR assay was used to screen a YAC library. Four positive cloneswere isolated. Subsequent characterization of these YACs byend-rescuing, restriction mapping, pulse field gel electrophoresis, andSouthern blots with the genetic crosses determined that two of theseYACs, adu and aad, were critical for subsequent studies. YAC aad is a550 kB nonchimeric YAC which extended furthest distally. Therefore, thedistal end of this YAC, aad(pICL) was used to complete the physical map.YAC adu is 370 kB nonchimeric YAC and its distal end, adu(+), wasdetermined to be nonrecombinant in all the ob progeny of the geneticcrosses including animals #111 and 167, suggesting that the ob genemight reside in this YAC.

A PCR assay for these two ends, aad(pICL) and adu(+) was developed andused for isolating moire YACs and P1 clones to continue physicalmapping. The important P1 clones isolated by this effort included 498,499, 500 (isolated using a probe derived from aad(pICL)) and 322, 323and 324 (using a probe from adu(+)).

In the meantime, YACs isolated by D6Rck13 (53A6, 25A8, 25A9, 25A10) werecharacterized. These studies determined that 53A6 extended furthestproximally toward the aad YAC. The size of the gap between 53A6 and aadwas determined ˜70 kB. The key end of 53A6, 53(pICL) was then used toscreen three available YAC libraries and a P1 library. A critical P1clone, 325, was isolated. This P1 clone overlapped with the P1 clonesisolated by aad(pICL) as described above, and therefore served to closethe gap between 53(pICL) and aad(pICL). As a result, the whole contig,containing YACs and P1 clones, of ˜2.5 million base pairs in length, andwhich spanned Pax4, 16M(+), adu(+), aad(pICL), 53(pICL), D6Rck39 andD6Rck13, was cloned. By carefully mapping the sites of recombinationapparent in animal #111 and 167, it was concluded that ob was situatedin a 400 kB interval. To provide a working DNA source for isolating theob gene, about 500 kB covering this nonrecombination region was isolatedin a total of 24 P1 clones. These P1 clones, including 322 and 323,which later were proved to be useful clones, were used for exontrapping.

The physical map of the portion of the chromosome carrying ob is shownin FIG. 7A. FIG. 7B represents the YAC contig. FIG. 7C represents the P1contig.

C. Isolation of Candidate Genes

The method used to isolate genes in this interval was exon trapping.This method used a commercial vector to identify exon DNA (i.e., codingsequences) by selecting for functional splice acceptor and donorsequences in genomic DNA introduced into a test construct. The DNA fromthese P1s were grown and subcloned into the exon trapping vector. Theseclones were short inserts cloned into a Bluescript vector. Each clonewas PCR amplified with PCR primers corresponding to plasmid sequencesthat flanked the insert. The PCR amplification was performed directly onthe bacteria that carried the plasmid. The reactions were set up using aBiomek robot. The PCR products were electrophoresed on a 1% agarose gelin TBE buffer that contained ethidium bromide. The exon trappingtechnique was modified to eliminate contaminating E. coli DNA from theP1 clones, and to screen out the abundant artifactual exons, whichexceeded 80-90% of the putative exons trapped. The exon trapping vectorincludes HIV sequences; a short segment of these vector sequencescorresponds to this artifact.

The exon trapping experiment was performed using various P1 clones. Exontrapping products were then amplified by PCR, selected, and sequenced.Sequences of putative “exons” were compared with those in Genbank usingthe Blast computer program. About 15 exons were selected for furtherexamination by RT-PCR, Northern analysis, and zoo blot for the presenceof corresponding RNA or conservative sequences. Seven of the 15 putativeexons, 325-2, 323-9, 322-5, D1-F7, 1H3, and 2G7, were found to encode anRNA transcript. 325-2 is a testis specific gene; 323-8 and 323-9 arelikely two exons from the same gene expressed mainly in brain andkidney. IH3 and 322-5 represent two low level brain transcripts. D1-F7is an exon from a previously cloned gene, inosine monophosphatedehydrogenase (IMPDH), which has ubiquitous expression pattern. None ofthese genes appeared to encode ob. 2G7, which is the ob exon, isdiscussed further below.

After three unsuccessful efforts to exon trap the ob gene, anotherattempt was made by pooling DNA from all the P1s from the critical obregion. These included P1s: 258, 259, 322, 323, 324, 325, 498, 499, 500,653, 654 and others. Thereafter P1s 258, 260, 322, 498 and 499 weresubcloned into the exon trapping vector, and subsequently several plateswere prepared with bacterial clones, each of which carried a putativeexon. Approximately 192 clones representing putative ob candidates wereobtained. As noted above, a consistent artifact such that many of theisolates contained two trapped exons derived from the vector wasobserved.

Thus, clones were identified both by their size and the fact thathybridization of DNA probes corresponding to this artifact hybridized tothe corresponding bands on a Southern blot of the gel. In this way, 185out of 192 clones were excluded from further evaluation. Exclusion ofthe artifacts on the basis of size alone was not possible, as this couldhave, in the end, led to exclusion of the exon corresponding to ob.

Thus, of the 192 exons, a total of seven exons were selected for furtherstudy. Templates for sequencing the seven exons were prepared, andsequencing was performed. The sequences for the 7 exons were analyzedand it was found that 4 were identical and one was an apparent artifact.In particular, clone 1D12 contained the “HIV sequence,” i.e., theartifact band. This left three exons for further analysis: 1F1, 2G7 and1H3. 1F1 was eliminated because it mapped outside the critical region.PCR primers for both 1 H3 and 2G7 were selected and synthesized.

The sequence of the exon on 2G7 was determined, and is shown in FIG. 10(SEQ ID NO:7). PCR primers for 2G7 were selected and synthesized. Theportions of the sequence corresponding to the PCR primers areunderlined. The primers used were:

5′ CCA GGG CAG GAA AAT GTG (SEQ ID NO: 8) (Tm = 60.0) 3′CAT CCT GGA CTT TCT GGA TAG G (SEQ ID NO: 9) (Tm = 60.0)

These primers amplified genome DNA with PCR conditions as follows: 25-30cycles at 55° annealing×2′, 72° extension×2′, 94° denaturation×1′ instandard PCR buffer. These primers were also used to generate a labeledprobe by including ³²P dCTP in the PCR reaction with a correspondingreduction in the amount of cold dCTP.

A RT PCR was performed on a variety of tissue RNAs and it was concludedthat 2G7 was expressed exclusively in white fat among the tissuesexamined (FIG. 11A). Thereafter, ³²P-labelled 2G7 was hybridized to aNorthern blot of tissue RNAs (FIG. 11B) and showed that its RNA wasexpressed at high level in fat tissue but was either not expressed orexpressed at very low levels in all other tissues (where the signals maybe the result of fat contaminating the tissue preparations). Ten μg oftotal RNA from each of the tissues listed was electrophoresed on anagarose gel with formaldehyde. The probe was hybridized at 65° in astandard hybridization buffer, Rapid Hype (Amersham). The size of theRNA was approximately 4.9 kB. At this point 2G7 was considered to be aviable candidate gene for ob and was analyzed further.

D. Mutation Detection

In order to confirm that 2G7 encoded the ob gene, it was necessary todemonstrate differences in the levels of RNA expression of DNA sequenceof this gene in mutant as compared to wild type animals. Two separatemutations of the ob gene are available for study, C57BL/6J ob/ob (1J)and Ckc/Smj ob/ob (2J). These will be referred hereinafter as 1J and 2J,respectively. (Informal nomenclature is used to refer to the mousestrains studied. Throughout this specification and in the drawings, itwill be understood that C57BL/6J refers to C57BL/6J+/+; CKC/smj refersto SM/Ckc−+^(Dac)−+/+; CKC/smj ob/ob refers toSM/Ckc−+^(Dac)−ob^(2J)/ob^(2J)). RNA was prepared from fat tissue thathad been isolated from 1J, 2J, and control animals. Total RNA for eachsample was treated with DNase and then reverse transcribed usingoligo-dT as a primer and reverse transcriptase. The resulting singlestranded cDNA was then PCR amplified either with the 2G7 primers(conditions shown above) for the lower band or commercially availableactin primers for the upper band. The RT PCR products were run on a 1%agarose TBE gel that was stained with ethidium bromide (FIG. 12A). UsingRT-PCT it was found that while 2G7 mRNA was expressed in 1J and all theother control mice, it was completely missing in 2J mouse. No signal wasdetected after 30 cycles of amplification. This experiment provideddirect evidence that 2G7 corresponded to an exon from the ob gene.

Since 2J mutation is relatively recent and is maintained as a coisogenicstrain, this result was the first available evidence that indicated that2G7 is an exon from the ob gene. The mutation is likely located in thepromoter region which leads to total abortion of the mRNA synthesis. Thepresence of signal in 1J mouse in this RT-PCT experiment suggested that1J might carry a point mutation which does not result in a gross changein size of the RNA sample. In addition, 2G7 mRNA was absent, when testedby RT PCR, from four additional 2J animals.

This result was confirmed on a Northern blot (FIG. 12B). Fat cell RNAwas prepared from each of the strains (C57Bl/6J, 1J, CKC/smj, and 2J).Ten μg of these RNAs were run out. The blot was probed with the 2G7probe that was PCR labeled, by amplification of the material, i.e.,band, in FIG. 11 using ³²P-dCTP in the PCR reaction. Actin is a controlfor the amount of RNA loaded. The actin signal is fairly similar in allof the samples. The ob signal is absent in brain because the mRNA isspecific to fat cells.

The results of the Northern analysis confirm that 2G7 RNA was absent in2J mice. The ob RNA is absent in the CKC/smj ob/ob mice because in thisobese mutant strain the gene is disrupted such that no RNA is made. Inaddition, the level of 2G7 RNA was increased ˜10-20 fold in 1J as wellas db/db fat. These results are compatible with the hypothesis that obeither encodes circulating hormone or is responsible for the generationof a signal from fat cells that modulates body weight. These resultssupported the conclusion that 2G7 is the ob gene and predicted that 1Jmice have a point mutation, probably a nonsense mutation leading to apremature translation termination.

These Northern results have been replicated using fat cell RNApreparations from four different 2J animals (FIG. 13). In this assay,ap2 is a fat-specific transcript that was used as a control much thesame as actin in FIG. 12B. There is no significance to the varyingdensity of the ap2 band. ap2 was labeled by designing PCR primers formthe published ap2 sequence. The RT PCR products of fat cell RNA werethen relabeled using the same protocol for PCR labeling. This analysisdemonstrates the presence of ob mRNA in normal homozygous orheterozygous animals, and its absence from 2J mutant animals.

The mutation has been identified in 1J mice. The mutation is a C to Tbase change that results in change of an arginine to an apparentpremature stop codon at amino acid 108, and in all likelihood accountsfor the 1J mutation (FIG. 14) despite high level expression of the obmRNA (see FIGS. 12 and 13, C57BL/6J ob/ob lanes).

More recently, Southern blots have been used to conclude that the 2Jmutation is the result of a detectable DNA change at the 5′ end of obthat appears to completely abolish RNA expression. The exact nature ofthis possible rearrangement remains to be determined.

A genomic Southern blot of DNA from the CKC/smj (SM/Ckc−+^(Dac)) andC57BL/6J mice using four different restriction endonucleases wasperformed in order to determine whether the mutant ob yielded a uniquefragment pattern (FIG. 15A). Approximately 10 μg of DNA (derived fromgenomic DNA prepared from liver, kidney, or spleen) was restrictiondigested with the restriction enzyme indicated. The DNA was thenelectrophoresed in a 1% agarose TBE gel. The DNA was transferred to animobilon membrane and hybridized to the PCR labeled 2G7 probe. The keyband is the uppermost band in the BglII digest for the CKC/smj ob/ob(SM/Ckc−+^(Dac)ob^(2J)/ob^(2J)) DNA. This band is of higher molecularweight than in the other strain, indicating a mutation in this strain.

FIG. 15B is a southern blot of a BglII digest of genomic DNA from theprogeny of an ob^(2J)/+×ob^(2J)/+cross. Some of the DNAs have only theupper band, some only the lower band, and some have the both bands. Theanimals with only the upper band are allo-obese, i.e., ob^(2J)/ob^(2J).These data show that the polymorphism (i.e., mutation) shown in FIG. 15Asegregates in a genetic sense.

cDNa Cloning and Sequence Determination of ob

Using the labeled 2G7 PCR probe, a total of 50 mouse cDNA clones from amurine fat cell λgt11 cDNA library (Clonetech 5′-STRETCH cDNA fromtesticular fat pads of Swiss mice, #ML3005b), and thirty crosshybridizing human cDNA clones from a human fat cell λgt10 cDNA library(Clonetech 5′-STRETCH cDNA from abdomen #HL1108a) were isolated. Libraryscreening was performed using the plaque lift procedure. The filtersfrom the plaque lift were denatured using the autoclave method. Thefilters were hybridized in duplicate with the PCR labeled 2G7 probe(Rapid Hybe buffer, 65° C., overnight). After a 2-4 hourprehybridization, the filters were washed in 2×SSC, 2% SDS, twice for 30minutes at 65° C. and exposed to SRy Llim. Duplicate positives wereplaque purified. Plaque purified phage were PCR amplified usingcommercially available vector primers, e.g., λ gt10 and λ gt11. Theresulting PCR products corresponded to the cDNA insert for each phagewith a small amount of vector sequence at either end. The bands were gelpurified and sequenced using the ABI automated sequencer and the vectorprimers to probe the DNA polymerase.

The raw sequencing data were then manually examined base by base tocorrect mishearing from the computer program. As the correct sequencebecame available, the downstream primers were synthesized and used tocontinue sequencing. Such experiments were repeated until each availablecDNA clone was sequenced and synthesized into a contig. To date, ˜3000base pairs from the 5′ end of the mRNA has been compiled. One of thecDNA clones extended to the 5′ end of the mRNA since its sequence wasidentical to that of the 5′ RACE product of fat tissue RNA (data notshown).

The sequence data revealed that there is a 167 amino acid open readingframe (FIG. 1). A Kozak translation initiation consensus sequence waspresent with an adenosine residue three bases upstream of the ATG. Twoclasses of cDNA were found differing by inclusion or exclusion of asingle glutamine codon. This residue is found in a position immediately3′ to the splice acceptor of the 2G7 exon. Since the CAG codon ofglutamine includes a possible AG splice acceptor sequence, it appearsthat there is slippage at the splice acceptor site with an apparent 3base pairs deletion in a subset of the cDNA, as shown below.

   gln ser val ag CAG TCG GTA (with glutamine) (SEQ ID NO: 16)    ↑(splice acceptor site)        ser val ag CAG TCG GTA (without glutamine)(SEQ ID NO: 17)        ↑ (splice acceptor site)

The “ag” in the sequences above corresponds to the assumed intronsequence upstream of the glutamine codon, and AG is the putativealternative splice site. This glutamine residue is located in a highlyconserved region of the molecule and its importance for biologicalactivity is as yet unknown.

A putative N-terminal signal sequence was detected, the signal cleavagesite of which is predicted to be carboxy terminal to the alanine residueat amino acid position 21. This putative signal sequence was confirmedby application of a computer algorithm to the method of von Heijne(Nucl. Acids Res. 14, 4683, 1986). Using this technique, the mostprobable signal sequence was identified in the polypeptide coding regioncorresponding to amino acids 1-23, having the sequence:

MCWRPLCRFLWLWSYLSYVQA ↑ VP (SEQ ID NO: 10)in which the arrow indicates the putative signal sequence cleavage site.The rest of the amino acid sequence was largely hydrophilic and did nothave any notable structural motifs or membrane spanning domains otherthan the N-terminal signal sequence. Specifically, we did not findconsensus sequences for N-linked glycosylation or dibase amino acidsequences indicative of protein cleavage in the predicted processedprotein (Sabatini and Adesnik, The metabolic basis of inherited disease,C. V. Scriver et al. eds., McGraw-Hill: New York, pp. 177-223). Database search using Blast and Block programs did not identify anyhomologous sequence.

Human fat tissue RNA was analyzed on Northern blot, RNA species ofsimilar size to the mouse ob gene was detected. Sequencing and analysisof cDNA clones revealed that human ob also encodes 167 amino acidpolypeptide (FIGS. 2 and 3). Two classes of cDNA with or without threebase pairs deletion were found in human as well (FIG. 6). The mouse andhuman ob genes were highly homologous in the predicted coding region,but had only 30% homology in the available 3′ and 5′ untranslatedregions. An N-terminal signal sequence was also present in the human obpolypeptide. Comparison of the human and mouse ob polypeptide sequencesshowed that the two molecules share an overall 84% identity at aminoacid level (FIG. 4). The N-termini of the mature proteins from bothspecies share even higher homology, with only four conservative andthree nonconservative amino acid substitutions among the N-terminal 100amino acid residues.

Genomic DNA was isolated from mouse, rat, rabbit, vole, cat, cow, sheep,pig, human, chicken, eel, and drosophila, and restriction digested withEcoRI. The digests were electrophoresed on 1% agarose TBE gel. DNA wastransferred to an imobilon membrane and probed with the PCR labeled 2G7probe. The filter was hybridized at 65° C. and washed with 2×SSC, 0.2%SDS at 65° C. twice for twenty minutes each wash, i.e., there were twobuffer changes. These data indicate that ob is conserved amongvertebrates (FIG. 16). Note in this regard that there is a 2+ signal ineel DNA; eel is a fish.

In summary, available evidence suggests that body weight and adiposityare physiologically controlled. Seven years ago efforts began toidentify two of the key components of this system: the ob and db genes.As shown in this example, the ob gene has now been identified as a fatspecific gene that plays a key role in regulating body weight. Theproduct of this gene, which is most probably a secreted hormone, willhave important implications for the diagnosis and treatment ofnutritional disorders in man and non-human animals.

Example Expression of ob in Bacteria

Both murine and human cDNAs encoding ob have been cloned into a pET-15bexpression vector (Novagen). This vector contains a T7 promoter inconjunction with a lac operator, and expresses a fusion proteincontaining a histidine tag (His-Tag) and a thrombin cleavage siteimmediately upstream of the coding sequence insertion site (FIG. 17)(SEQ ID No:11).

The mouse and human cDNAs were modified such that the alanine at the endof the signal sequence was turned into an NdeI site, as was a separatesequence in the 3′ region. Insertion of the NdeI site was accomplishedusing PCR with novel primers:

Mnde-5′ (murine five prime primer): CTTATGTTCA TATGGTGCCG ATCCAGAAAG TC(SEQ ID NO: 12) Mnde-3′ (murine three prime primer):TCCCTCTACA TATGTCTTGG GAGCCTGGTG GC (SEQ ID NO: 13) Hnde-5′(human five prime primer): TCTATGTCCA TATGGTGCCG ATCCAAAAAG TC(SEQ ID NO: 14) Hnde-3′ (human three prime primer):TTCCTTCCCA TATGGTACTC CTTGCAGGAA GA (SEQ ID NO: 15)

The primers contain a 6-base pair mismatch in the middle that introducesNdeI restriction sites at each end of the PCR fragment. Phage carryingeither the mouse or human cDNA were PCR amplified using those primers.The PCR product was digested with NdeI and gel purified on a 1% lowmelting point agarose gel. The gel purified bands were subcloned intothe pET vector. The resulting plasmids were sequenced to ensure thatmutations were not introduced during the PCR amplification step ofcloning. Constructs for the human and murine cDNA that encodes and thatlacks glutamine 49 have been prepared. In particular, pET 15b constructscontaining either the human or the mouse ob coding sequence, minussignal sequence and fused to a Hig-Tag, have been made using a PCRcloning method. The constructs have been sequenced to ensure no sequenceerrors were introduced into the coding region of the ob gene during thePCR amplification step.

Two resultant plasmid constructs, pETM9 and pETH14, were selected totransform a bacterial expression host. Upon induction with 1 mM IPTGunder optimal conditions, the transformed bacteria were able to produce100-300 μg/ml of the ob fusion. The majority of the ob fusion proteinwas found in the inclusion body. After solubilization with 6Mguanidine-HCl or urea, the fusion protein was purified through aHis-binding (Ni-chelation) resin column. The conditions for columnpurification of the ob fusion protein (including binding, washing, andeluting) were established experimentally. The ob fusion protein binds tothe resin at 5 mM imidazol/6M guanidine-HCl and stays bound at up to 20mM imidazol/6M guanidine-HCl. The protein can be eluted form the resinat 60 mM imidazol/6M guanidine (FIG. 18A,B). Both the purified human andmouse ob fusion proteins were further dialyzed in PBS to removeguanidine-HCl from the preparation and used to raise polyclonalantibodies.

In order to test the biological activity of the fusion protein products,the refolding conditions for the purified protein was tested anddeveloped. This involves initial dialysis of the fusion protein in 1 Mguanidine solution, followed by dilution with 0.4 M arginine solution.The His-Tag was removed from the fusion proteins before biologicalfunction assay. The tag removal was achieved by treating the fusionprotein with thrombin from human placenta.

In addition, human and mouse ob gene coding sequence minus the signalsequence is being inserted into a pET 12c vector using PCR cloningmethod. These constructs can direct the synthesized ob fusion proteinsinto the periplasmic space of the bacterial host cell. The ob fusionprotein recovered from the periplasmic space may only need a simple gelfiltration to be purified from other host proteins and will not bedenatured during such process.

Example Preparation of Antibodies to the ob Polypeptide

In addition to use of the recombinant protein to generate polyclonalantibodies, a set of four peptide sequences from the deduced murine obsequence were identified using immunogenicity plot software (GCGPackage). The four carboxyl terminal peptide fragments are:

(SEQ ID NO: 18): Val-Pro-Ile-Gln-Lys-Val-Gln-Asp-Asp-Thr-Lys-Thr-Leu-Ile-Lys-Thr (SEQ ID NO: 19):Leu-His-Pro-Ile-Leu-Ser-Leu-Ser-Lys-Met-Asp-Gln- Thr-Leu-Ala(SEQ ID NO: 20): Ser-Lys-Ser-Cys-Ser-Leu-Pro-Gln-Thr-Ser-Gly-Leu-Gln-Lys-Pro-Glu-Ser-Leu-Asp (SEQ ID NO: 21):Ser-Arg-Leu-Gln-Gly-Ser-Leu-Gln-Asp-Ile-Leu-Gln-Gln-Leu-Asp-Val-Ser-Pro-Glu-Cys

These peptides were conjugated to KLH, and the peptide-KLH conjugateswere used to immunize rabbits using standard techniques. Polyclonalantisera specific for each peptide is recovered from the rabbits.

Example In Vitro Translocation of an ob Polypeptide

In order to confirm the presence of a functional signal sequence, ahuman cDNA that included the entire open reading frame was subclonedinto the pGEM vector.

Only the human cDNA was used in this experiment because suitable mousesubclones were not recovered. Positive strand human ob RNA wastranscribed using sp6 polymerase and used in an in vitro translationreaction with and without canine pancreatic microsomal membranes. Theprimary translation product migrated with an apparent molecular weightof ˜18 kD, which is consistent with that predicted by the cDNA sequence.Inclusion of the microsomal membranes in the reaction inhibited theoverall efficiency of translation ˜5 fold. Nevertheless, approximately50-70% of the ob primary translation product was truncated byapproximately 2 kD in the presence of the membrane preparation,suggesting that the signal sequence is functional (FIG. 19A). The sizeof the primary translation product of interleukin-1α RNA, which does notencode a signal sequence, was unchanged when microsomal membranes wereincluded in the reaction. In order to confirm that translocation of theob protein had taken place, the in vitro translation products weretreated with Proteinase-K. Protease treatment resulted in the completeproteolysis of the 18 kD primary translation product while the 16 kDprocessed form was unaffected by the enzyme treatment, indicating thatit had translocated into the lumen of the microsomes (FIG. 19B). Thesedata are compatible with the hypothesis that ob is a secreted molecule.

After signal sequence cleavage, two cysteine residues would remainwithin the predicted protein raising the possibility that the moleculecontains a disulfide bond characteristic of other secreted polypeptides(Shen and Rutter, 1984, Science 224:168-171).

Example Characterization of the ob Gene

To establish the relationship between obesity and genetic alterations inthe ob gene in humans, the sequence of the human ob gene was determined(FIG. 20A) (SEQ ID NO:). Specific primers from the human coding sequencewere used to screen a human P1 library. Three different P1 clones wereobtained, grown up, and PCR amplified using primers flanking thesplicing site between the first and second coding exon. The entireintron region, around 2 kB, was amplified and partially sequenced (seeFIG. 20A; and as indicated in SEQ ID NO:22).

The gene structure of both the murine and human genes was characterizedusing PCR assays and other standard techniques. The mouse ob gene wasfound to consist of 3 exons, the second and third of which account forthe coding sequence (FIG. 20B). The coding region of the human ob geneshares the same structure; however, the human gene lacks a 5′ exon andintron (FIG. 20C).

Two sets of primers generated from the intronic sequences of the humangene have been prepared (FIG. 20A). The sequences of the primers follows(F and R refer to forward and reverse, respectively):

HOB 1gF 5′-CCCAAGAAGCCCATCCTG-3′ (SEQ ID NO: 26) HOB 1gR5′-GACTATCTGGGTCCAGTGCC-3′ (SEQ ID NO: 27) HOB 2gF5′-CCACATGCTGAGCACTTGTT-3′ (SEQ ID NO: 28) HOB 2gR5′-CTTCAATCCTGGAGATACCTGG-3′ (SEQ ID NO: 29)

DNA samples have been obtained from various sources, and these sets ofprimers are being used to amplify human genomic DNA from severely obesepeople. The PCR products were run on a low melting point agarose gel,and the bands were cut out and digested with agarase. The sequences wereobtained using the ABI 373A DNA sequencer and Taq dideoxy terminator kit(abi, Perkin-Elmer). One point mutation in an ob gene from a patientsample has been detected to date. This mutation is on the first exon anddoes not change the amino acid sequence. Preliminary data indicate thatan insertion sequence may be present in the first exon of anotherpatient.

A different automated sequencing method with Sequenase instead of TaqDNA polymerase may be employed to yield more easily readable sequencesfor mutation detection.

Example Expression of ob in Yeast

Following the positional cloning of ob, it became important to uncoverthe physiological mechanism by which the ob protein reduces food intakeand body weight. The first step in this direction was to recombinantlyproduce a functional protein using an expression system. In addition tothe successful bacterial expression system, a yeast expression systemwas also selected. Yeast expression has several attractive features forexpressing ob. The most important is that biologically active eukaryoticproteins are more likely to be produced. The ob polypeptide is secretedby mammalian cells. Protein secretion is very similar for alleukaryotes, which means that the yeast secretory apparatus is much moresimilar to the mammalian secretory pathway than bacterial secretorypathways would be. In particular, protein modifications of ob seen inmammalian cells would likely also be seen in the expression through theyeast secretory system. In addition, protein folding is carried out inpassage through the secretory apparatus and thus delivering ob throughthe yeast secretory apparatus is likely to give a properly foldedprotein with native biological activity. This is significant for obbecause the two cystein residues may form a disulfide bridge. Incontrast to secretory pathways, the reducing environment of the cellcytoplasm prevents formation of disulfide bridges, and therefore it isessential that ob pass through the secretory pathway in order for thisdisulfide bond to form in vivo. Other advantages have to do with theease and quickness of manipulating yeast, the availability of vectorsand strains, and the vast experience in yeast recombinant technology.

A Pichia pastoris expression system was chosen for four reasons: (1) ithas higher levels of heterologous protein expression than other yeastsystems such as S. cerevisiae; (2) protein glycosylation is more similarto the mammalian system in P. pastoris than S. cerevisiae (althoughglycosylation sites were not detected in ob using a computer search,there still remained the possibility of glycosylation at unrecognizedsites); (3) P. pastoris secretes very few proteins natively, and thus itis generally straightforward to purify the expressed foreign protein;and (4) the vectors and yeast strains are commercially available (fromInvitrogen). Two strategies for generating yeast expression vectors areshown in FIGS. 21 and 22.

The vector chosen was pPIC.9. This vector contains a cloning site justdownstream of the alpha-mating factor prepro coding sequence whichdirects the protein encoded by the gene cloned into the cloning site tobe secreted by the secretory pathway. The other important feature of thevector is a HIS4 gene that allows selection for uptake of the vectorusing a yeast auxotrophic strain grown on histidine-deficient mediafollowing transformation of the yeast with the vector. The cloningstrategy was the following: PCR amplify ob cDNA using a 5′ primer thatcontained at its 3′ end sequence complementary to the sequence of objust following the predicted leader peptide cleavage site, and at itsmost end a sequence complementary to the 3′ end of the alpha-matingfactor sequence of the vector. The 5′ primer also contains an XhoI site.The 3′ primer was designed to have at its 3′ end a sequencecomplementary to the last few amino acids of ob and an EcoRI site at its5′ end. Following PCR amplification, the PCR product was digested withXhoI and EcoRI and cloned into similarly digested pPIC.9. Following thecloning of both the mouse and human ob cDNAs, each with and without theglutamine at codon 49, individual clones were isolated for all fourindividual constructs and sequenced to verify that the constructs werecloned in the correct orientation and frame and contained no mutationsfrom the PCR amplification step. Following identification of clones withthe correct sequence, these were transformed into P. pastoris strainGS115, a histidine auxotroph.

For the two mouse ob constructs, transformed yeast clones were screenedfor protein expression. As evidence that the transformed yeast containob, a DNA dot-blot assay and a colony hybridization assay were donewhich both showed ob sequence within the transformed yeast but notwithin the untransformed yeast. Furthermore, the transformed yeast nowsecreted a 16 kDa protein into the culture media whereas theuntransformed yeast does not secrete a protein of this size (FIG. 23A).This is the predicted size of ob. Individual clones for both mouseconstructs have been identified that are high expressors for ob, andcurrently a purification strategy is being developed to purify ob tohomogeneity. One strategy has been to purify ob on a cation exchangecolumn (FIG. 23B); preliminary data suggest that a strong cationexchanger may be useful. However, after cation exchange chromatography,the putative ob product is lost. This indicates the presence of aprotease in the sample.

One strategy to overcome this problem is to prepare ob-His tag fusionsfor expression in yeast (FIG. 22). Further evaluation has demonstratedthat ob without a His tag associates tightly with a Ni-chelation column.Purification of the ob polypeptide by Ni-chelation, followed by gelfiltration, yielded a product of sufficient purity for mass spectralanalysis. Mass spec confirms the molecular weight of the expressedprotein is identical to the expected molecular weight, which stronglyconfirms that ob has been successfully expressed in Pichia.

However, the Ni-chelation/gel filtration purification protocol does notyield a ob polypeptide in sufficiently pure form. Additional smallmolecules are present. It does appear that the proteolytic activityelutes from the Ni-chelation column in the void volume. Accordingly, athree step purification process is planned: Ni-chelation, followed bycation exchange (which eliminates the small molecule contaminants),followed by gel filtration.

Estimating expression level by Coomassie blue staining of SDS-PAGE gelsreveals approximately 10 mg/L when yeast are grown in shaker flasks.These levels are expected to increase in fermentation vessels, and weare about to initiate fermentation with the hopes of obtaining largerquantities of protein. Regarding the human ob constructs, transformedyeast clones containing high copy numbers of the ob gene have beenidentified, and these are expected to express ob protein. As antibodiesare developed, these will be used to confirm the identity of thesecreted 16 kDa protein.

Once purified the expressed protein is characterized by several methods.Physical characterization includes light-scattering to determinehomogeneity of structure and is used as a measure of proper folding.Circular dichroism is used to roughly determine the structural geometryof the protein. Importantly, bioactivity of the purified protein isassayed by administering the protein to both elan and obese rodents viaan osmotic pump (e.g., an ALZET osmotic pump from Alza Corporation, PaloAlto, Calif.) over at least a two-week period and observing effects onfeeding behavior and body weight.

The following is a list of references' related to the above disclosureand particularly to the experimental procedures and discussions.

-   Bahary, N.; G. Zorich; J. D. Pachter; R. L. Leibel; and J. M.    Friedman. 1991. Molecular genetic linkage maps of mouse chromosomes    4 and 6. Genomics 11:33-47.-   Bahary, N.; D. McGraw; R. L. Leibel; and J. M. Friedman. 1991.    Chromosomal microdissection of midmouse chromosome 4: Mapping of    microclones relative to the mouse db gene. Submitted.-   Bahary, N.; J. Pachter; R. Felman; R. L. Leibel; K. A. Albright; S.    Cram; and J. M. Friedman. 1991. Molecular mapping of mouse    chromosomes 4 and 6: Use of a flow-sorted Robertsonian chromosome.    Submitted.-   Blank, R.; J. Eppig; F. T. Fiedorek; W. N. Frankel; J. M.    Friedman; K. Huppi; I. Jackson; and B. Mock. 1991. Mouse chromosome    4. Mammalian Genome 1 (suppl): s51-s78.-   Bogardus, C.; Ravussin, E.; Abbot, W.; Zasakzku, J. K.; Young, A.;    Knowler, W. C.; Friedman, J. M.; R. L. Leibel; N. Bahary; D. A.    Siegel; and G. Truett, G. 1991. Genetic analysis of complex    disorders: Molecular mapping of obesity genes in mice and humans.    Annals of the New York Academy of Sciences 630:100-115.-   Friedman, J. M.; R. L. Leibel; and N. Bahary. 1991. Molecular    mapping of obesity genes. Mammalian Genome 1:130-144.-   Friedman, J. M.; R. L. Leibel; N. Bahary; and G. Zorich. 1991.    Molecular mapping of the mouse ob mutation. Genomics, (in press).-   Harris, M. I. (1991). Diabetes Care 14 (suppl. 3), 639-648.-   Harris, M. I.; Hadden, W. C.; Knowler, W. C.; and Bennett, P. H.    (1987). Diabetes 36, 523-534.-   Harris, R. B. S. (1990). FASEB J. 4, 3310-3318.-   Jacobowitz, R., and Moll, P. O. (1986). N. Engl. J. Med. 315, 96-100-   Kessey, R. E. (1980). In Obesity, A. Stunkard, eds.    (Philadelphia: W. B. Sauders Co.), pp. 144-166.-   Kessey, R. E., and Pawley, T. L. (1986). Annu. Rev. Psychol. 37,    109-133.22-   Leibel, R. L., N. Bahary and J. M. Friedman. 1990. Genetic variation    and nutrition in obesity: Approaches to the molecular genetics of    obesity. In Genetic variation and Nutrition (Simopoulos, A. P. and    Childs, B., eds.), S. Karger, Basel, pp. 90-101.-   Siegel, D.; N. G. Irving; J. M. Friedman; and B. J.    Wainwright. 1991. Localization of the cystic fibrosis transmembrane    conductance regulator to mouse chromosome 6. Cytogenetics Cell    Genetics, submitted.-   Truett, G. E.; N. Bahary; J. M. Friedman; and R. L. Leibel. 1991.    The rat obesity fatty (fa) maps to chromosome 5: Evidence for    homology with the mouse gene diabetes (db). Proc. Natl. Acad. Sci.    USA 88:7806-7809.

Example Weight Reducing Effects of the Ob Polypeptide (Leptin)

(Various references are cited by author, year, and # in this Example,which citations correlate with the list of references found at the endof this Example.)

The gene product of the mouse ob locus plays an important role inregulating body weight. We establish that the ob protein circulates inmouse, rat and human plasma. The circulating form in all three specieshas an identical molecular weight by SDS-PAGE to the deduced polypeptidesequence without the signal sequence, suggesting that in vivo theprotein is not processed after cleavage of the signal sequence. The obprotein is absent in plasma from C57/Bl6J ob/ob mice and is present atten-fold higher concentrations in plasma of db/db mice and twenty-foldhigher levels in plasma of fa/fa rats relative to controls. These obeseanimal mutants have been suggested to be resistant to its effects. Therewere seven fold differences in plasma levels of the ob protein within agroup of six lean human subjects. Daily injections of the recombinantmouse ob protein dramatically reduce body mass in ob/ob mice, havesignificant effects on body weight of wild type mice but have no effecton db/db mice. These data show that the gene product of the ob locusserves an endocrine function to regulate body weight. We propose thatthe protein encoded by the ob gene be named Leptin derived from theGreek root lephós meaning thin.

Materials and Methods

Rabbits were immunized with recombinant protein in Freunds adjuvant.(HRP, Inc) Immunopurified anti-mouse ob antibodies were prepared bypassage of antiserum over a sepharose 4B column conjugated to therecombinant protein as described[Harlow, 1988 #444]. Immunoprecipitationof mouse plasma was carried out as follows. 0.5 ml of plasma from mouse,rat and human containing approximately 2.5 mM EDTA was pre-cleared withunconjugated sepharose-4B at room temperature with rocking for 2 hours.The sepharose was removed by spinning and 50 μl added of a 50% slurry ofantibody-conjugated sepharose containing affinity purified antibody at aconcentration of 1 mg/ml of packed sepharose. 0.5 ml of 2×RIPA bufferwas added to give final binding conditions as follows: 50 mM Tris-HCl,pH7.5, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and0.025% sodium azide. The reaction was carried out overnight at 4° C.with rocking. The antibody-conjugated sepharose was washed 8 times withRIPA buffer and rinsed three times with PBS and run on 15% SDS-PAGE. Theproteins were transferred to nitrocellulose and Western blotted with abiotinylated immunopurified antibody against the recombinant protein Thesecondary used was HRP-streptavidin and ECL was used for detection.

To quantitate the amount of ob in mouse serum, increasing amounts of therefolded recombinant mouse ob protein (0.01, 0.1, 0.5, 2.0, 15.0 ng) wasadded to 100λ of C57BL/6J ob/ob plasma and incubated at 4° C. for 3hours with the protein A sepharose conjugated antibody. After extensivewashing with buffer A (10 mM Na Phosphate buffer, pH 7.4; 100 mM NaCl;1% Triton X-100, 5 mM EDTA, 1 mM PMSF) samples were resuspended insample buffer and loaded on a 15% SDS-PAGE and transferred to anitrocellulose membrane. Western blotting was performed using animmunopurified biotinylated anti-amino terminus antibody as a primaryantibody and HRP-Streptavidin as a secondary antibody followed by ECLdetection.

Cytoplasmic extracts were prepared by homogenizing adipose tissue in NDSbuffer by polytron and dounce homogenization and removal of nuclei bycentrifuging at 700 g. [10 mM Tris, pH 7.5, 10 mM NaCl, 60 mM ICCI, 0.15mM spermine, 0.5 mM spermidine, 14 mM β-Mercaptoethanol, 0.5 m EGTA, 2mM EDTA, 0.5% NP-40]

Immunoprecipitations were performed as above except that immunopurifiedanti-human ob antibodies were used. For the ELISA, 100 μl of a 1 μg/mlsolution of immunopurified anti-human ob antibody was dissolved in aborate buffered PBS solution and applied overnight to microtiter(Corning cat. #2595) plates at 4° C. The plates were then washed 4 timeswith borate saline solution containing 0.05% Tween 20 and excess liquidwas removed. Plates were blocked by incubation at RT for 2 hours with240 μl per well of borate saline buffer containing 0.3% gelatin and thenwashed and dried. Either known amounts of a refolded human ob protein orplasma samples in 100 μl volume were incubated in individual wellsovernight at 4° C. After washing, the plates were incubated with 100 μlof a biotinylated immunopurified anti-human antibody (0.1 mg/ml in agelatine borate buffered solution) for 4 hours at room temperature.After washing Horse Radish Peroxidase-Streptavidin was added to theplates (0.1 μg/ml in borate buffer, 0.3% gelatin). HRP substratesolution (ABTS, 0.3 mg/ml and H₂O₂, 0.01% in citric acid) was then usedfor detection and the OD at 414 nM was read to quantitate the antibodybinding.

The mouse and human ob coding sequence were PCR amplified from plasmidscontaining ob cDNA sequences and subcloned into the pPIC.9 plasmid(Invitrogen). The human 5′ primer was: 5′GTATCTCTCGAGAAAAGAGTGCCCATCCAAAAAGTCCAAG 3′ and the 3′ primer was 5′GCGCGAATTCTCAGCACCCAGGGCTGAGGTC 3′. For mouse the 5′ primer was: 5′GTATCTCTCGAGAAAAGAGTGCCTATCCAGAAAGTCCAGG 3′ and the ‘3’ primer was 5′GCGCGAATTCTCAGCATTCAGGGCTAACATC 3′. The 5′ primer for both mouse andhuman contains an XhoI site at the 5′ end and coding sequences for thelast 4 aa of the alpha-mating factor signal sequence present in thevector pPIC.9. This vector directs secretion of heterologously expressedgenes from the cell into the culture media. The 5′ PCR primer alsoincludes the first 19 n.t.'s of the ob open reading frame after thesignal sequence cleavage site before the alanine at amino acid position21. The 3′ primer contains an EcoRI site at it's 5′ end which isimmediately followed by sequences complementary to the putative ob stopcodon. The PCR conditions were as follows: denaturing for 1′ at. 94,annealing for 1′ at 55 and extension for 2.5° at 72. Low-cycle PCR (15cycles) and the proof-reading polymerase PFU (Stratagene) were used tolimit the number of PCR-generated mutations. The PCR products weredigested with XhoI and EcoRI and cloned into similarly digested vectorpPIC.9. All constructs were sequenced on both strands to ensure theabsence of any PCR-generated mutations. Clones were transformed intoPichia pastoris (His-) by the spheroplast method and selected onhistidine deficient media. Approximately 200 clones of mouse and humanwere screened for high-copy number integration by a colony hybridizationassay and the high copy number clones were then assayed for obexpression initially by coomassie staining showing the presence of anovel 16 kd protein present in the culture media of transformed yeast.The 16 kd band was confirmed to be ob using antibodies raised againstthe bacterially expressed protein. The recombinant proteins werepurified by a two-step purification method described below. Massspectrometry and cyanogen bromide treatment were performed asdescribed[Beavis, 1990 #804].

The entire ob coding sequence of the mouse and human ob genes C-terminalto the signal sequence were subcloned into the Pet15b expression vector(Novagen) and overexpressed in Escherichia coli [BL21(DE3)pIYsS] usingthe T7 RNA polymerase system[Studier, 1990 #803]. Cells grown at 30° C.to an absorbency of 0.7 at 595 nM and induced with 0.5 mMisopropyl-β-D-thiogalcto-pyranoside overnight were collected bylow-speed centrifugation. Lysis was performed by three cycles of freezethaw and DNA digestion was done with DNasel. Membrane extraction wasperformed by sonication and detergent solubilization, and the finalinclusion body pellet was dissolved in 6M guanidine-HCl, 20 mM HEPES,pH8.4. Recombinant ob proteins were purified under denaturing conditionsby IMAC using a Ni-ion affinity column and washing with increasingamounts of imidazole. Purified denatured ob protein was then stored in 6M guanidine-HCl, 10 mM sodium acetate (NaAc), pH5, and reduced using 1mM DTT at room temperature for 1 hour. Denaturation was performed bydiluting the reduced protein into 20% glycerol, 5 mM CaCl₂, 5 mM NaAc,pH5, through mixing and incubation at room temperature for 8-12 hours.After denaturation the pH was adjusted to 8.4 by addition of Tris to 10mM, and the hexahistidine tag was removed by thrombin cleavage. Cleaved,renatured protein was repurified by IMAC to separate product fromthrombin and uncleaved fusion protein. Cleaved, renatured protein elutesfrom the Ni-ion affinity column at 40 mM imidazole, whereas thrombin isnot retained and uncleaved fusion protein elutes at 0.2 mM imidazole.Product was then concentrated, treated with 100 mM EDTA and 10 mMpotassium ferricyanide and further purified by gel filtration usingPharmacia superdex 75 16/60 column.

An Ellman's assay was conducted as described[Ellman, 1959 #798].Ellman's reagent was prepared by dissolving 39.6 mg5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) in 10 ml 0.05 M phosphate,pH 8. A calibration curve was constructed in the concentration range of10-120 mM free sulfhydryl (using a 1 mM stock solution of reduced DTT)at 412 nm. Each assay was performed using 0.02 ml Ellman's reagent and atotal reaction mixture of 0.5 ml. The measured extinction coefficientwas 12974 M⁻¹ cm⁻¹ for free sulfhydryl group (correlation coefficient0.99987), which is within 5% of the previously reported value of 13600M⁻¹ cm⁻¹.

50 μl of 2 μg/ml pure gel filtered protein, corresponding to a possiblefree sulfhydryl concentration of about 24 μM in the final reactionmixture, was subjected to Ellman's assay. The resulting solution gaveA₄₁₂ of about 0.02, suggesting that two cysteine residues in the proteinare in oxidized state to form cystine or that their free sulfhydrylgroups are completely buried within the inaccessible core of the foldedprotein. Identical results were obtained by conducting the same assay onunfolded protein in the presence of 6 M guanidine-HCl.

Mice were individually caged in a pathogen-free environment andacclimated to a diet containing 35% (w/w) Laboratory Rodent Diet 5001(PMP Feeds, Inc.), 5.9% (w/w) tapioca pudding mix (General Foods) and59.1% water which has an energy content of 1.30 kcal/gm. The diet wassterilized by autoclave and packed into 60 mm plastic dishes which werefixed to the tops of 100 mm petri dishes. Tapioca gives the diet a pastytexture making it difficult for the animal to spread the food in thecage. The 100 mm lid recovers the small amount of food spilled by theanimal. A fresh dish of food was placed into the cage each morning andthe previous day's dish was removed and weighed. The difference inweight provided a measure of daily food consumption. Effects ofrecombinant protein on food intake and body weight were measured inthree strains of mice: C57Bl/6J ob/ob, C57 Bl/Ks db/db and CBA/J +/+,purchased from the Jackson Laboratory. Thirty mice from each strain weredivided into groups of 10. One group from each strain received dailyintraperitoneal (i.p.) injections of the refolded bacterial ob proteinat a dose of 5 μg/g/day in 300 μl of PBS A second group received i.p.injections of the same volume of PBS. These control mice receivedinjections of the PBS dialysate of the recombinant protein. The PBS wascleared of endotoxin using an Acticlean ETOX column. A third group ofanimals did not receive injections. Food intake was recorded daily andbody weight measurements were recorded regularly over a 3.5 weekinterval. For the pair feeding experiment, the food intake of a separategroup of ob mice was matched on a daily basis to that consumed by the obmice receiving protein.

Results

The ob Protein Circulates in Mouse, Rat and Human Plasma

Recombinant mouse and human ob protein was prepared using the PET 15bbacterial expression vector (Novagen) and by cloning into Pichiapastoris, a yeast expression system that secretes recombinant proteinsdirectly into the culture media. The ob protein expressed in yeastincludes the 146 amino acids carboxy terminal to the signal sequence.Rabbits were immunized with the bacterial proteins (HRP, Inc.).Antibodies were immunopurified (Research Genetics) and used forimmunoprecipitations and Western blots of protein from plasma andadipose tissue.

The ob protein from mouse plasma migrates with an apparent molecularweight of 16 kD by SDS-PAGE. The electrophoretic mobility is identicalto the recombinant ob protein secreted by yeast after signal sequenceremoval. (FIG. 24A) The protein was not detected in plasma from C57BL/6Job/ob mice that have a nonsense mutation at codon 105. Several differentantisera failed to identify the truncated 105 residue polypeptide chainpredicted by the cDNA sequence.

A ten-fold increase in the level of circulating protein was observed indb/db mice relative to a control animal (FIG. 24A). Immunoprecipitationof plasma from wild type and fa/fa rats revealed a twenty-fold increasein the level of the ob protein in the mutant rat compared to wild type.(FIG. 24B) The db mutation results in an obese phenotype identical tothat seen in ob mice[Bahary, 1990 #31]. fatty, rats are obese as aresult of a recessive mutation in a gene homologous to db[Truett, 1991#409]. In order to quantitate the level of ob in mouse plasma,increasing amounts of recombinant protein were added to ob serum andimmunoprecipitated. (FIG. 24C) A linear increase of the signal intensityon Western blots was seen with increasing amounts of recombinantprotein. Comparison of the signal intensity of the native protein inmouse plasma to the standards indicated that the circulating level ofthe ob protein in wild type mice is ˜20 ng/ml. These data demonstratethat the immunoprecipitations and Western blots were performed underconditions of antibody excess. Increased levels of the ob protein werealso seen in protein extracts of adipose tissue from db/db mice relativeto controls. (FIG. 24D) As expected for a secreted protein, the proteinfrom the adipose tissue fractionated with the crude membrane fraction(data not shown).

Plasma samples from six lean human subjects with a Body Mass Index lessthan 25 (BMI=weight/length²) were immunoprecipitated usingimmunopurified antibodies to the human protein. The immunoprecipitatedmaterial migrated with an electrophrotic mobility identical to that seenfor the 146 amino acid human protein expressed in yeast. The intensityof the signals varied significantly among the six samples. (FIG. 25A)Densitometry of the autoradiograph revealed an ˜five-fold difference inthe levels in individuals HP1 and HP6 with intermediate levels in theother subjects. An enzyme linked immunoassay (ELISA) was developed usingthe immunopurified antibody and the refolded bacterial protein as astandard (see below). The resulting standard curve is shown in FIG. 25B.Using this assay, the plasma levels of the ob protein in the six humanplasma samples varied between 2-15 ng/ml. (FIG. 25C) The level of the obprotein in plasma from HP 6 was outside of the linear range of theimmunoassay and is ≧ or 15 ng/ml. These quantitative differencescorrelated with those seen on Western blots.

Structural Features of the ob Protein

As the ob protein has two cysteine residues, it could form either intra-or intermolecular disulphide bonds under oxidizing conditions in vivo.Western blots were repeated with and without the addition of reducingagents to the sample buffer. Under both conditions, the ob protein inhuman serum migrated as a monomer (data not shown). Under nonreducingconditions, protein immunoprecipitated from db mouse serum was detectedat positions consistent with that of both a monomer of 16 kD and a dimerof ˜32 kD. (FIG. 26A) The higher molecular weight moiety disappearedunder reducing conditions suggesting that a fraction of mouse obcirculates as a higher molecular weight species via formation of anintermolecular disulphide bond. Approximately 80% of mouse ob circulatesas the ˜16 kD protein and 20% as the ˜32 kD form.

The same molecular forms are seen when the mouse and human proteins areexpressed in Pichia pastoris [Abrams, 1992 #708]. In these studies, theDNA sequence corresponding to the 146 amino acid mature ob protein wascloned downstream of the yeast alpha mating factor signal sequence inthe pPIC.9 vector (Invitrogen). ob protein was purified from the yeastmedia of strains expressing the mouse and human proteins andelectrophoresed under reducing and nonreducing conditions. (FIG. 26A)The mouse protein was expressed in yeast mainly as a dimer undernonreducing conditions, and only as a monomer in the presence ofreducing agents. The recombinant human protein migrated to the positionof a monomer under both conditions (data not shown).

The purified human protein expressed in Pichia had a molecular mass of16,024±3 Da as determined by mass spectrometry[Beavis, 1990 #804]. Thisvalue is in agreement with the mass calculated from the amino acidsequence of the protein containing a single intramolecular disulfidebridge (16,024 Da). Matrix-assisted laser desorption mass spectometricanalysis of cyanogen bromide cleavage products of the protein indicatesthat cysteines 117 and 167 are linked through an intramoleculardisulphide bond. (FIG. 26B) Cyanogen bromide; cleaves carboxyterminal tomethionine residues.

Preparation and Characterization of Bioactive Recombinant Protein

Mouse ob protein was expressed in E. coli. from a PET 15b plasmid as aninsoluble fusion protein, with a twenty residue, N-terminalhexahistidine tag containing a thrombin cleavage site. Bacterialinclusion bodies were solubilized using guanidine-HCl and purified underdenaturing conditions using immobilized metal ion affinitychromatography (IMAC). (FIG. 27) Purified, denatured fusion protein wasreduced, diluted and permitted to refold in aqueous solution at roomtemperature. Following thrombin cleavage, renatured mouse ob proteincontaining four additional N-terminal residues (Gly-Ser-His-Met) wasrepurified by IMAC to >98% homogeneity, as judged by SDS-PAGE and massspectrometry. Matrix-assisted laser desorption mass spectrometry gave ameasured mass of 16,414±3 Da (predicted mass=16,415 Da). Both reducingand non-reducing SDS-PAGE gels demonstrated a single molecular specieswith apparent and molecular weight of 16 kD (data not shown).

Dynamic light scattering using a DP801 Molecular Size Detector (ProteinSolutions, Inc.) demonstrated that the renatured mouse ob protein waslargely monomeric, with some higher-order aggregates. The protein wastreated with EDTA and chemically oxidized. Higher molecular weightspecies were then removed by gel filtration.

Further dynamic light scattering confirmed that the purified, renaturedrecombinant mouse ob protein was monodispersed. Following dialysisagainst phosphate buffered saline (PBS), bacterial endotoxin was removedusing an Acticlean ETOX column (Sterogene Bioseparations, Inc.). Thefinal yield of protein was 45 mg/l.

Ellman's assay was performed on the purified, renatured recombinantmouse ob protein to assess its oxidation state[Ellman, 1959 #798]. Bothrenatured protein and protein unfolded by 6M guanidine-HCl demonstrated<0.5% free sulfhydryl content, demonstrating that the monomeric productcontains an intramolecular disulphide bond. This was confirmed by massspectrometry of the cyanogen bromide cleavage products of the refoldedbacterial protein (data not shown),

Bioactivity of the ob Protein

The purified, renatured recombinant mouse ob protein was administered asa daily intraperitoneal injection of 5 mg/kg/day to groups of 10C57Bl/6J ob/ob (age, 16 weeks), C57Bl/Ks db/db (age, 12 weeks) andCBA/J+/+ (age, 8 weeks) mice. An equal number of animals received PBS asa daily injection. The PBS used for the control injections was derivedfrom the dialysate after equilibration of the protein. Ten additionalanimals from the three mouse strains did not receive injections. Thefood intake of individual animals was monitored daily and the weights ofthe animals were recorded at three or four day intervals. The cumulativeresults for food intake and body weight from each of the 9 groups ofmice are shown in FIG. 28A and the statistical significance of the dataare shown in Table 1. The food intake of the C57Bl6J ob/ob mice injectedwith protein was significantly decreased after the first injection andcontinued to decrease until the fifth day when it stabilized at a levelequal to ˜40% of the intake of the animals receiving injections of PBS(p<0.001). The sham injected ob mice did not lose weight over the threeweek study period. The C57Bl/6J ob/ob mice receiving protein lostapproximately 10% of their body weight after 5 days (p<0.001). Theseanimals continued to lose weight over the three week treatment at whichpoint the weight of the ob animals receiving protein had decreased to anaverage of 60% of their initial body weight (p<0.0001). A separate groupof ob mice were pair fed to the ob mice receiving protein. The data inFIG. 28B show that the pair fed mice lost significantly less weight thanthe animals receiving the recombinant protein. (p<0.02). A photograph oftwo mice receiving injections of either protein or vehicle shows thegross difference in appearance resulting from the protein treatment(FIG. 28C). In order to further ascertain the effects of the protein,autopsies of two mice in each of the groups were performed. Grossinspection of the ob mice receiving protein revealed a dramatic decreasein body fat as well as the size of the liver. The liver weights of thedb and wild type mice were unchanged with treatment. The livers from theob mice receiving the injections of PBS weighed 5.04 and 5.02 grams vs.2.23 and 2.03 grams in the animals receiving the recombinant protein. Incontrast to the pale fatty liver characteristic of ob mice, the liverfrom the ob mice receiving protein acquired the darker colorcharacteristic of normal liver (FIG. 28D). Histologic sections of theliver indicated that the untreated animals had a fatty liver that wasmarkedly improved by protein treatment (data not shown).

In contrast to the ob mice, there were no significant differences inbody weight or food intake in the C57BL/Ks db/db mice receiving proteinrelative to the control group receiving vehicle. (FIG. 28A, Table 1) Allthree groups of db/db mice lost between 2-5 grams during the treatmentperiod. The average blood glucose of the db mice was measured using aglucometer, and was ≧500 mg/dl in all of the mice indicating that theseanimals had developed diabetes secondary to obesity. The injections ofdb mice were terminated after two weeks.

In wild type mice there was a small but significant decrease in bodyweight following administration of the recombinant ob protein. (FIG.28A, Table 1). After five days of protein injection, the treated micelost an average of 0.5 grams while control mice gained 0.4 grams(p<0.02). At two subsequent time points the animals receiving proteinweighed significantly less than the mice receiving daily injections ofPBS. The significance of the weight change was reduced at the later timepoints. In the animals that lost weight, the food intake was notsignificantly different from control animals. The injections of PBS hada small but significant effect on food intake and body weight in ob, dband wild type mice as compared to mice not receiving injections(p<0.05).

TABLE 1 Animal Treatment WEIGHT CHANGE Group Group Days n Mean Std.Error p ob/ob protein 1-5  10 −6.38000000 0.47628190 <0.001 vehicle 9−0.14444444 0.24444444 protein 1-12 10 −14.45000000 0.70793126 <0.001vehicle 9 0.98888889 0.38058597 protein 1-27 6 −24.28333333 0.69924563<0.0001 vehicle 5 4.30000000 0.79874902 db/db protein 1-5  10−1.47000000 0.36939891 0.240 vehicle 10 −2.00000000 0.23142073 protein1-12 10 −3.75000000 0.77348418 0.610 vehicle 10 −4.19000000 0.34655447CBA/J protein 1-5  10 −0.48000000 0.17876117 0.006 vehicle 10 0.380000000.21489015 protein 1-12 10 −0.12000000 0.45748103 0.015 vehicle 101.20000000 0.18378732 protein 1-27 5 1.98000000 0.48723711 >0.651vehicle 6 2.23333333 0.20763215

The effects of protein vs PBS injections on body weight are tabulatedfor C57Bl/6J ob/ob, C57Bl/Ks db/db and wild type mice. Shown are theaverage values, standard errors and statistical significance at each ofthe time points listed. Four animals in the CBA/J and ob groups and allof the db mice were sacrificed after two weeks of injections.Statistical significance was determined using a two sample of test.

Discussion

An endocrine function for the protein product of the ob locus was firstsuggested by Coleman, who showed that the body weight of ob/ob mice wasreduced after parabiotic union to normal or db mice[Coleman, 1978 #42].Our results support this hypothesis by showing that ob proteincirculates in the bloodstream and that injections of recombinant proteinreduce body weight. The molecular weight of the gene product encoded bythe ob gene is ˜16 kD, which is equal to the 146 amino acid sequencecarboxy terminal to the signal sequence. The recombinant ob protein isnot modified when expressed in Pichia pastoris. Expression of mammaliangenes in Pichia generally results in the formation of the correctprotein structure[Cregg, 1993 #792]. These findings suggest that the obprotein is not glycosylated and is not post-translationally processed invivo. The data do not exclude the possibility that the ob protein isnoncovalently bound to itself or other proteins in plasma or adiposetissue. Although proteolytic cleavage of the protein has not beenexcluded, lower molecular weight forms of the ob protein were notdetected by any of the antisera used, including four anti-peptideantibodies.

The ob protein has two cysteine residues and circulates as a monomer inhuman, and as a monomer and dimer in mouse. An intramolecular disulphidebond typical of secreted molecules is found when the human protein isexpressed in Pichia pastoris suggesting that it is likely to be presentin vivo. This is supported by the bioactivity of the recombinantbacterial protein, which has an intramolecular disulphide bond. Themouse ob protein can be found in plasma as a monomer and as a dimer.That monomer and dimer are seen when the mouse ob protein is expressedin yeast shows that the propensity of the mouse protein to form a dimeris a result of differences in the primary sequence relative to human.While it is clear that the monomer has bioactivity, the functionalactivity of the dimer is unknown.

The effect of the ob protein on food intake and body weight in ob miceis dramatic. After three weeks treatment, the ob mice receiving dailyinjections of recombinant protein had lost 40% of their weight and wereconsuming 40% as much food as control animals. Moreover, the weight ofthe treated ob mice had not yet equilibrated at the time the experimentwas terminated. The results of the pair feeding experiment indicateweight loss is a result of effects on both food intake and energyexpenditure. Thus, a separate group of ob mice whose caloric intake wasrestricted to that of ob mice receiving protein lost significantly lessweight than the animals receiving protein. The reduction in food intakein ob/ob mice to a level lower than that of wild type mice, within a dayof receiving the ob protein, indicates that they are especiallysensitive to its effects. Indeed, the ob receptor may be upregulated inthese animals. Food intake of treated ob mice became relatively constantafter five days of treatment. If this is the result of the proteinhaving reached steady state levels, it would suggest that the proteinhas a relatively long half life[Goodman, 1990 #793]. This conclusion isconsistent with data from parabiosis experiments[Coleman, 1978 #42;Weigle, 1988 #349].

Effects of recombinant protein on the body weight of wild type mice weresmall but statistically significant during the first two weeks of thestudy. While the difference in weight between wild type mice receivingprotein vs. PBS was sustained at later time points, the statisticalsignificance of the data had greatly diminished after three weeks. Theearly weight loss could not be accounted for by a difference in foodintake. Presumably, the measurement of food intake was not preciseenough to detect a decrease resulting in a one gram difference in bodyweight during treatment. These observations differ from the results ofprevious experiments in which wild type rodents have been joined byparabiotic union to db mice, fatty rats, rats with hypothalamic lesionsand rats rendered obese by a high calorie diet[Coleman, 1978 #42;Harris, 1987 #800; Harris, 1989 #799; Hervey, 1959 #305]. In each case,the wild type animals become anorectic and lose copious amounts ofweight. As the levels of ob protein are increased in db mice and fa ratsand the level of ob RNA is increased in mice with hypothalamic lesions,it is likely that wild type mice can respond to ob when it circulates inplasma at a sufficiently high level. The findings reported here areconsistent with the possibility that the levels of the administeredprotein were below endogenous levels, leading to equilibration at aslightly lower body weight. Quantitation of the circulating levels ofthe ob protein in the treated mice will resolve this issue.Immunoprecipitations have suggested that the levels c circulating obprotein were not substantially elevated in the wild type mice receivingprotein

The lesser effect of the protein on wild type mice and the absence of aresponse in db mice makes it unlikely that the treatment has nonspecificor aversive effects. All of the db mice lost a small amount of weightduring the treatment period, whether or not they were receiving the obprotein. The db animals were markedly hyperglycemic and the weight lossis likely to be the result of diabetes and not the experimentalprotocol. C57BL/Ks db/db mice often develop diabetes and begin to losesmall amounts of weight when of the age of the animals used in thisstudy[Coleman, 1978 #42]. C57Bl/6J ob/ob mice of a similar age do notdevelop significant hyperglycemia. These phenotypic differences arethought to be the result of genetic differences in the strains (C57Bl6Jvs. C57Bl/Ks) carrying the mutations.[Coleman, 1978 #42].

The failure to detect the truncated 105 amino acid protein predicted bythe cDNA sequence of the ob gene in C57Bl/6J ob/ob mice suggests thatthe mutant protein is either degraded or not translated. However, thepossibility that the antisera used do not detect this truncated proteincannot be excluded. The observed ten-fold increase in the levels of theob protein in db mice compared to wild type suggests that the ob proteinis overproduced when there is resistance to its effects. These datacorrelate with studies of the ob mRNA (Next Example). As mentioned,previous experiments have shown that mutations of the mouse db and therat fa genes, which map to homologous chromosomal regions, result inoverproduction of a plasma factor that suppresses body weight[Truett,1991 #409; Coleman, 1978 #42; Hervey, 1959 #305]. In both cases, it hasbeen suggested that the mutant animals are resistant to the effects ofthe ob protein. This possibility is confirmed by the observation thatthe ob protein has no effect on body weight or food intake whenadministered to db mice.

Obesity in humans could be associated with increased levels of the obprotein in plasma in individuals who are relatively unresponsive to thehormone. On the other hand, reduced expression of ob could also lead toobesity in which case “normal” (i.e; inappropriately low) levels of theprotein might be found. Thus, the levels of ob protein in human plasmacould be a marker for different forms of obesity. In a small group oflean subjects with BMI<25, low nanogram levels of circulating ob proteinare detectable by ELISA. Significantly, variable concentrations werenoted suggesting that the level of expression and/or sensitivity to theprotein may play a role in determining body weight.

The site of action of the ob protein is unknown. The protein affectsboth food intake and energy expenditure, a finding consistent withclinical studies indicating that alterations of both systems act toregulate body weight[Leibel, 1995 #795; Keesey, 1984 #796]. Thehypothalamus is likely to be downstream of ob in the pathway thatcontrols body weight, although direct effects on a variety of organs arepossible

REFERENCES FOR THIS EXAMPLE

-   1. Kennedy, G. C. The role of depot fat in the hypothalamic control    of food intake in the rat. Proc. Roy. Soc. (London) (B)., 1953. 140:    578-592.-   2. Leibelt, R. A., S. Ichinoe, and N. Nicholson. Regulatory    influences of adipose tissue on food intake and body weight. Ann.    N.Y. Acad. Sci., 1965. 131: 559-582.-   3. Zhang, Y., P. Proenca, M. Maffei, M. Barone, L. Leopold,    and J. M. Friedman. Positional cloning of the mouse obese gene and    its human homologue. Nature., 1994. 372: 425-432.-   4. Maffei, M., H. Fei, G. W. Lee, C. Dani, P. Leroy, Y. Zhang, R.    Proenca, R. Negrel, G. Ailhaud, and J. M. Friedman. Increased    expression in adipocytes of ob RNA in mice with lesions of the    hypothalamus and with mutations at the db locus. (Next Example)-   5. Bahary, N., R. L. Leibel, L. Joseph, and J. M. Friedman.    Molecular mapping of the mouse db mutation. Proc. Nat. Acad. Sci.    USA., 1990. 87: 8642-8646.-   6. Truett, G. E., N. Bahary, J. M. Friedman, and R. L. Leibel. The    rat obesity gene fatty (fa) maps to chromosome 5: Evidence for    homology with the mouse gene diabetes (db). Proc. Natl. Acad. Sci.    USA., 1991. 88: 7806-7809.-   7. Abrams, J. S., M.-G. Roncarolo, H. Yssel, U. Andersson, G. J.    Gleich, and J. E. Silver. Strategies of anti-cytokine monoclonal    antibody development: Immunoassay of IL-10 and IL-5 in clinical    samples. Immunological Reviews., 1992.5-24.-   8. Ellman, G. L. Arch. Biochem. Biophy., 1959. 82: 70-77.-   9. Coleman, D. L. Obese and Diabetes: two mutant genes causing    diabetes-obesity syndromes in mice. Diabetologia., 1978. 14:    141-148.-   10. Cregg, J. M., T. S. Vedvick, and W. C. Raschke. Recent advances    in the expression of foreign genes in Pichia pastoris.    Bio/Technology., 1993. 11: 905-914.-   11. Goodman, L. S., in The Pharmacological Basis of    Therapeutics., A. Gilman, 1990, Pergamon Press: New York. p. 19-45.-   12. Weigle, D. S. Contribution of decreased body mass to diminished    termic/effect of exercise in reduced-obese men. Int. J.    Obesity., 1988. 12: 567-578.-   13. Harris, R. B. S., E. Hervey, G. R. Hervey, and G. Tobin. Body    composition of lean and obese zucker rats in parabiosis. Int. J.    Obes., 1987. 11: 275-283.-   14. Harris, R. B. S, and R. J. Martin, Physiological and metabolic    changes in parabiotic partners of obese rats., in Hormones,    Thermogenesis and Obesity, H. Lardy and F. Straatman, 1989, Elsevier    Science Publishing Co.: New York.-   15. Hervey, G. R. The effects of lesions in the hypothalamus in    parabiotic rats. J. Physiol., 1959. 145: 336-352.-   16. Leibel, R. L., M. Rosenbaum, and J. Hirsch. Changes in energy    expenditure resulting from altered body weight. The New England    Journal of Medicine., 1995. 332: 621-628.-   17. Keesey, R. C. and S. W. Corbett, Metabolic defense of the body    weight set-point, in Association for Research in Nervous and Mental    Disease, A. J. Stunkard and E. Stellar, 1984, Raven Press: New    York. p. 87-96.

Example Increased Expression in Adipocytes of Mice of ob mRNA Due toLesions of Hypothalamus and with Mutations at the db Locus

(Various references are cited by author, year, and # in this Example,which citations correlate with the list of references found at the endof this Example.)

The gene product of the recently cloned mouse obese gene (ob)) plays animportant role in regulating the adipose tissue mass. ob RNA isexpressed specifically by mouse adipocytes in vivo in each of severaldifferent fat cell depots including brown fat. It is also expressed incultured 3T3-442A preadipocyte cells that have been induced todifferentiate. Mice with lesions of the hypothalamus, as well as micemutant at the db locus, express a twenty-fold higher level of ob RNA inadipose tissue. These data suggest that both the db gene and thehypothalamus are downstream of the ob gene in the pathway thatregulates' the adipose tissue mass and are consistent with previousexperiments suggesting that the db locus encodes the ob receptor. In thedb/db and lesioned mice, quantitative differences in the level ofexpression of ob RNA correlated with the lipid content of adipocytes.The molecules that regulate the level of expression of the ob gene inadipocytes are likely to play an important role in determining bodyweight as are the molecules that mediate the effects of ob at its siteof action.

Materials and Methods

In Situ Hybridization

White fat tissues from identical abdominal regions of wild type (wt) anddb mice were processed simultaneously according to the modified methoddescribed by Richardson et al[6]. Briefly, tissues were fixed in Bouin'ssolution for 2 hours at 4° C. They were then dehydrated by serialtreatment of increasing concentrations of ethanol from 10% to 100%, eachfor 5 min. at 4° C. Further incubation of tissues with xylene (1h) andparaffin (2h) were performed at 65° C. Embedded wt and db/db fat tissueswere sectioned and mounted on to the samme conditions later. Sectionswere baked at 65° C. for 1 h and treated with xylene and serialdilutions of ethanol from 100% to 50%, each for 3 min. at roomtemperature. Antisense RNA probe of ob gene was synthesized by in vitrotranscription of linearized ob gene coding sequence upstream of a Sp6RNA polymerase promoter. In situ hybridization was carried out exactlyaccording to Schaeren-Wiemers and Gerfin-Moser[7].

RNA Preparation and Cell Culture

Total RNA and Northern blots were prepared as described[3]. Stromalvascular cells and adipocytes were prepared according to Rodbell and RNAfrom both fractions was prepared according to Dani et al[8, 9]. Aftersub-cloning, 3T3-F442 cells were grown in Dulbecco's modified Eaglemedium containing 10% foetal bovine serum (defined as standard medium)[10]. At confluence, cells were treated in standard medium supplementedwith 2 nM triiodothyronine (T3) and 17 nM insulin. Twelve days later,RNA was prepared as above.

Gold ThioGlucose Treatment

Two month old female CBA/J mice were treated with a singleintraperitoneal injection of aurothioglucose (Sigma A0632) at a dose of0.2 mg/g in normal saline. Control animals were injected with normalsaline. Mice were weighed one month after the treatment. Adipose tissueRNA was isolated from those treated animals whose weight had increasedmore that twenty grams post GTG treatment.

Results

The ob gene was recently found to be expressed in adipose tissue[3]. Asadipose tissue is composed of many cell types including adipocytes,preadipocytes, fibroblasts and vascular cells, in situ hybridization wasperformed to sections of epididymal fat pads from normal animals withsense and antisense ob riboprobes[6, 11]. When using the antisenseprobe, positive signals were detectable in all of the adipocytes in thesection (FIG. 29—labeled Wt). Signals were not noted when the antisenseprobe was hybridized to sections of brain (data not shown).Hybridization of the antisense probe to sections of adipose tissue fromC57Bl/Ks db/db mice was greatly increased, confirming the adipocytespecific expression of ob RNA and demonstrating a large increase in thelevel of ob RNA per adipocyte in these animals (FIG. 29—labeled db/db).Mice mutant at the db locus are massively obese as part of a syndromethat is phenotypically identical to that seen in C57Bl/6J ob/obmice[12].

ob RNA was not synthesized by adipose tissue stromal cells separatedfrom adipocytes. As expected, cells in the adipocyte fraction expressedob RNA using Northern blots (FIG. 30). The same result was obtainedusing RT-PCR (data not shown). These data support the conclusion thatonly adipocytes express the ob gene. Data from cultured adipocytesconfirm this conclusion. In these studies, 3T3-F442A cells were culturedusing conditions that lead to lipid accumilation, as part of a cellularprogram leading to differentication into adipocytes. ob RNA was notexpressed in exponentially growing cells as well as in confluent3T3-F442A preadipocyte cells which express early markers whiledifferentiation of these cells into adipocytes led to the expression ofdefectable levels of ob RNA (FIG. 30)[13]. The level of ob RNA isextremely sensitive to the culture conditions as no message was observedin late post-confluent cells not exposed to insulin.

Hybridization studies showed that ob RNA is expressed in vivo in severaldifferent fat depots including the epididymal, parametrial, abdominal,perirenal, and inguinal fat pads (FIG. 31A). The precise level ofexpression in each of the depots was somewhat variable, with inguinaland parametrial fat expressing lower levels of ob RNA. ob RNA is alsoexpressed in brown adipose tissue although the level of expression is˜50 fold lower in brown fat relative to the other adipose tissue depots.These quantitative differences correlated loosely with previouslyreported differences in cell size among the different fat celldepots[14]. The amount of ob RNA in brown fat is unaffected by coldexposure (FIG. 31B). In this experiment, the level of uncoupling proteinRNA (UCP) increased in brown fat after cold exposure while the level ofob RNA did not change[15]. In aggregate, these data confirm that alladipocytes are capable of producing ob RNA and demonstrate a variablelevel of expression in different fat depots. These data support thepossibility that the level of the encoded protein correlates with thetotal adipose tissue mass.

We next measured the levels of ob RNA in db/db mice and mice withlesions of the hypothalamus. Lesions of the ventromedial hypothalamus(VMH) result in obesity as part of a syndrome resembling that seen inob/ob and db/db mice[16]. Parabiosis experiments suggest such lesionsresult in over expression of a blood borne factor that suppresses foodintake and body weight[17]. Similar results are noted when mice mutantat the db locus are parabiosed to normal mice, suggesting the obreceptor may be encoded by the db locus[18]. Thus, obesity resultingfrom VMH lesions and the db mutation may be the result of resistance tothe effects of the ob protein. If so, a secondary increase in the levelsof ob RNA in adipose tissue would be predicted.

Hypothalamic lesions were induced in female CBA mice using the chemicalGold ThioGlucose (GTG)[19]. This treatment results in specifichypothalamic lesions, principally in the ventromedial hypothalamus(VMH), with the subsequent development of obesity within several weeks(manuscript in preparation). In our experience, a single intraperitonealinjection of GTG of 0.2 mg/gm body weight results in the development ofobesity within four weeks. One month old female CBA/J mice (20-25 grams)were treated with GTG and the subsequent weight gain of treated andcontrol animals is shown. (Table 2) Adipose tissue RNA was prepared fromdb/db mice and from those GTG treated animals that gained >20 gm.Northern blots showed a twenty-fold increase in the level of ob RNA intwo month old db/db and GTG treated mice compared to normal animals(FIG. 32).

TABLE 2 Weight Gain in Gold ThioGlucose Treated Mice control (n = 41)GTG (n = 93) <10 g  41, (100%) 4, (4%) 10 g-20 g 0, (0%) 15, (16%) >20 g0, (0%) 74, (80%)

Two month old female CBA/J mice were treated with goldthioglucose (GTG).Goldthioglucose (Sigma A0632) was administered intraperitonealy innormal saline solution at a dosage of 2.0 mg/g. Body weight of controland injected animals was recorded before and one month after theinjection. Animals were housed five to a cage and were fed ad libitum.The amount of weight gained one month postinjection is shown in thetable. Animals with a body weight gain greater that 20 g one month afterinjection were selected for further study.

Discussion

The gene product of the mouse ob gene circulates in mouse and humanplasma where it may act to regulate the adipose tissue mass (manuscriptin preparation). Further studies on the regulation of expression andmechanism of action of ob will have important implications for ourunderstanding of the physiologic pathway that regulates body weight.

In this report we show that the ob gene product is expressed exclusivelyby adipocytes in all adipose tissue depots. This result is consistentwith the possibility that the protein product of the ob gene correlateswith the bodies lipid stores. Moreover ob RNA is upregulated twenty foldin db mice and mice with hypothalamic lesions. In these animals, theactual increase in the level of ob RNA per cell is likely to be evenhigher than twenty fold since the adipocyte cell size is increased ˜fivefold in these animals (see FIG. 29) [14]. These data position the dbgene and the hypothalamus downstream of ob in the pathway that controlsbody weight and is consistent with the hypothesis that the ob receptoris encoded at the db locus[18]. The molecular cloning of the ob receptorand/or the db gene will resolve this issue. The increase in the level ofob RNA in db/db and GTG treated mice also suggests a non cell-autonomousfunction of the ob gene product in fat cells[4, 5]. Thus, if the encodedprotein acted directly on fat cells to inhibit growth ordifferentiation, the overexpression of the wild type ob gene in GTGtreated mice would result in a lean phenotype.

The most parsimonious explanation of these data is that the ob proteinfunctions as an endocrine signaling molecule that is secreted byadipocytes and acts, directly or indirectly, on the hypothalamus. Directeffects on the hypothalamus would require that mechanisms exist to allowpassage of the ob gene product across the blood brain barrier.Mechanisms involving the circumventricular organ and/or specifictransporters could permit brain access of a molecule the size of thatencoded by the ob gene[20-22]. However, this hypothesis must beconsidered with caution until the means by which the protein might crossthe blood brain barrier have been identified. Moreover, possible effectson other target organs will need to be evaluated.

The fat cell signal(s) that are responsible for the quantitativevariation in the expression level of the ob gene is not yet known butcorrelates with differences in adipocyte cell size. Adipocytes fromdb/db mice are five times as large as those from normal mice, with acell size of −1.0 μg lipid/cell[14]. Prior evidence has indicated thatfat cell lipid content and/or size is an important parameter indetermining body weight[23, 24]. It could be that each fat cellexpresses a low level of ob RNA that further increases in proportion tothe cell size. It is also possible that cell size is not the sensedparameter and merely correlates with the intracellular signal thatincreases the expression of the ob gene in adipocytes from db/db and VMHlesioned mice. In any case, the components of the signal transductionpathway regulating the synthesis of ob RNA are likely to be important indetermining body weight. Genetic and environmental influences thatreduce the level of expression of ob would act to increase body weightas would influences that decreased sensitivity to the encoded protein.The specific molecules that regulate the level of expression levels ofthe ob gene are as yet unknown, and await a determination of thelevel(s) of gene control that leads to quantitative variation in thelevel of ob RNA and an examination of the regulatory elements of the obgene. The identification of the molecules that regulate the expressionof the ob gene in adipocytes and those that mediate the effects of theencoded protein at its site(s) of action will greatly enhance ourunderstanding of the physiologic mechanisms that regulate body weight.

REFERENCES FOR THIS EXAMPLE

-   1. Kennedy, G. C. The role of depot fat in the hypothalamic control    of food intake in the rat. Proc. Roy. Soc. (London) (B), 1953. 140:    578-592.-   2. Leibelt, R. A., S. Ichinoe, and N. Nicholson. Regulatory    influences of adipose tissue on food intake and body weight. Ann.    N.Y. Acad. Sci, 1965. 131: 559-582.-   3. Zhang, Y., P. Proenca, M. Maffei, M. Barone, L. Leopold,    and J. M. Friedman. Positional cloning of the mouse obese gene and    its human homologue. Nature, 1994. 372: 425-432.-   4. Ashwell, M., C. J. Meade, P. Medawar, and C. Sowter. Adipose    tissue: contributions of nature and nurture to the obesity of an    obese mutant mouse (ob/ob). Proc. R. Soc. Lond., 1977. 195: 343-353.-   5. Ashwell, M. and C. J. Meade. Obesity: Do fat cells from    genetically obese mice (C57BL/6J ob/ob) have an innate capacity for    increased fat storage? Diabetologia, 1978. 15: 465-470.-   6. Richardson, R. L., J. T. Wright, J.-W. Kim, and G. J. Hausman.    Expression of transforming growth factor-β (TGF-(β1) and    insulin-like growth factor II (IGF-II) messenger RNA in the    developing subcutaneous tissue (SQ) of the fetal pig. Growth,    Development & Aging, 1992. 56: 149-157.-   7. Schaeren, N. and A. Gerfin-Moser. A single protocol to detect    transcripts of various types and expression levels in neural tissue    and cultural cells: in situ hybridization using digoxigenin-labeled    cRNA probes. Histochemistry, 1993. 100: 431-440.-   8. Rodbell, M. Metabolism of isolated fat cells. J. Biological    Chemistry, 1964. 239: 375-380.-   9. Dani, C., A. Doglio, A. Pradines-Figueres, and P. Grimaldi,    Molecular biology techniques in the study of adipocyte    differentiation., in Obesity in Europe: 88, P. Bjorntorp and R.    Rossner, Editor^Editors. 1989, John Libbey Company Ltd.: London,    England. p. 371-376.

10. Dani, C., et al. Regulation of gene expression by insulin in adiposecells: opposite effects on adipsin and glycerophosphate dehydrogenasegenes. Mol. Cell. Endocrinol., 1989. 63: 199-208.

-   11. Wasserman, M., The concept of the fat organ: in Rodahl,    Issekutz, fat as a tissue, in Rodahl, Issekutz, Fat as a tissue.    1964, McGraw Hill: New York. p. 22-92.-   12. Bahary, N., R. L. Leibel, L. Joseph, and J. M. Friedman.    Molecular mapping of the mouse db mutation. Proc. Nat. Acad. Sci.    USA, 1990. 87: 8642-8646.-   13. Dani, C. A., A. Doglio, E. Z. Amri, S. Bardon, P. Fort, B.    Bertrand, P. Grimaldi, and G. Ailhaud. Cloning and regulation of a    mRNA specifically expressed in the preadipse state. J. Biol.    Chem., 1989. 264: 10119-10125.-   14. Johnson, P. R. and J. Hirsch. Cellularity of adipose depots in    six strains of genetically obese mice. Journal of Lipid    Research, 1972. 13: 2-11.-   15. Jacobsson, A., U. Stadker, M. A. Glotzer, and L. P. Kozak.    Mitochondrial uncoupling protein from mouse brown fat. J. Biol.    Chem., 1985. 260: 16250-16254.-   16. Bray, G. A. and L. A. Campfield. Metabolic factors in the    control of energy stores. Metabolism, 1975. 24: 99-117.-   17. Hervey, G. R. The effects of lesions in the hypothalamus in    parabiotic rats. J. Physiol., 1959. 145: 336-352.-   18. Coleman, D. L. Obese and Diabetes: two mutant genes causing    diabetes-obesity syndromes in mice. Diabetologia, 1978. 14: 141-148.-   19. Debons, A. F., I. Krimsky, M. L. Maayan, K. Fani, and F. A.    Jimenez. Gold thioglucose obesity syndrome. Fed. Proc., 1977. 36:    143-147.-   20. Johnson, A. K. and P. M. Gross. Sensory circumventricular organs    and brain homeostatic pathways. FASEB Journal, 1983. 7: 678-686.-   21. Baura, G. D., D. M. Foster, D. Porte Jr., S. E. Kahn, R. N.    Bergman, C. Cobelli, and M. W. Schwartz. Saturable transport of    insulin from plasma into the central nervous system of dogs in vivo.    Jr. of Clinical Investigation, Inc., 1993. 92: 1824-1830.-   22. Pardridge, W. M. Receptor-mediated peptide transport through the    blood-brain barrier. Endocrine Reviews, 1986. 7: 314-330.-   23. Faust, I. M., P. R. Johnson, J. S. Stern, and J. Hirsch.    Diet-induced adipocyte number increase in adult rats: a new model of    obesity. Am. Journal of Physiol., 1978. 235: E279-86.-   24. Faust, I. M., P. R. Johnson, and J. Hirsch. Surgical removal of    adipose tissue alters feeding behavior and the development of    obesity in rats. Science, 1977. 197: 393-396.

Example RNA Expression Pattern and Mapping of the Human ob Gene on thePhysical, Cytogenetic, and Genetic Maps of Chromosome 7

(Various references are cited by author, year, and # in this Example,which citations correlate with the list of references found at the endof this Example.)

OB RNA is expressed at high levels in human adipose tissue and atsubstantially lower levels in placenta and heart. The human OB gene mapsto a large YAC contig derived from chromosome 7q31.3. In addition toconfirming the relative location of the gene based on mouse-humancomparative mapping, this study has identified 8 establishedmicrosatellite markers in close physical proximity to the human OB gene.Since mutations in mouse ob can result in a syndrome that closelyresembles morbid obesity in humans, these genetic markers representimportant tools for studying the possible role of the OB gene ininherited forms of human obesity.

Abbreviations

FISH: fluorescence in situ hybridization; GDB: Genome Data Base; kb:kilobase pairs; Mb: megabase pairs; PCR: polymerase chain reaction; STS:sequence-tagged site; YAC: yeast artificial chromosome.

Material and Methods

Northern Blot Analysis

Total RNA was prepared from adipose tissue using the method of Chirgwinet al. (1979). Northern blots, radiolabelling, and hybridizations wereperformed as described (Zhang et al. 1994). Northern blots of polyA+ RNA[human MTN, human MTN II, and human fetal MTN II] were purchased fromCLONTECH (Palo Alto, Calif.), as were PCR primers used to generate theradiolabelled human actin probe.

STS Development

STS-specific PCR assays were developed and optimized essentially asdescribed (Green and Green, 1991a, Green et al. 1991b, Green, 1993;Green et al. 1994). Each STS is named using the prefix sWSS followed bya unique number. Details about the 19 STSs reported here are provided inTable 1, with additional information (e.g., PCR reaction conditions,complete DNA sequence) available in GenBank and/or the Genome Data Base(GDB). For the microsatellite-specific STSs, the oligonucleotide primersused in the PCR assays (Table 3) corresponded either to those employedfor genotype analysis (Table 4) or those designed [most often with thecomputer program OSP (Hillier and Green, 1991)] using the DNA sequenceavailable in GenBank™.

TABLE 3 STSs in the YAC contig containing the human OB geneThe 19 chromosome 7-specific STSs mapped to the YAC contig containing the human OB gene(FIG. 34) are listed. In each case, the designated sWSS name, relevant alias, GDB-assigned locusname, STS source, PCR primer sequences, STS size, and GDB identification number areindicated. The sources of STSs are as follows: YAC End [isolated insert end of a YAC (Green,1993)], Lambda Clone [random chromosome 7-specific lambda clone (Green et al. 1991b; Green,1993)], Genetic Marker [microsatellite marker (Green et al. 1994), see Table 2], YAC Insert[random segment from YAC insert], and Gene [gene-specific STS]. Note that for some geneticmarker-specific STSs, the PCR primers used for identifying YACs (listed in this table) aredifferent from those used for performing genotype analysis (Table 4), since the detection ofYACs containing a genetic marker does not require amplification of the polymorphic tract itself.All of the indicated PCR assays utilized an annealing temperature of 55°C., except for sWSS494, sWSS883, sWSS1529, and sWSS2619 (which used 50°C.), sWSS999 and sWSS1174 (which used 60°C.), and sWSS808 (which used 65°C.). Additional details regarding the STS-specific PCRassays are available in GDB. Size STS Name Alias Locus SourcePCR Primers (bp) GDB ID No. sWSS1734 D7S2185 YAC EndCAAGACAAATGAGATAAGGAGAGTTACAGCTTTA 72 G00-455-235 CAG sWSS494 D7S2016Lambda  CTAAACACCTTTCCATTCCTTATATTCACTTTTC 112 G00-334-404 Clone CCCTCTCsWSS883 UT528 D7S1498 Genetic  TGCAGTAAGCTGTGATTGAGGTGCAGCTTTAATT 490G00-455-262 Marker GTGAGC sWSS2359 AFMa065zg9 D7S1873 Genetic AGTGTTGTGTTTCTCCTGAAAGGGGATGTGATAA 142 G00-455-247 Marker GTG sWSS2336AFMa125wh1 D7S1874 Genetic  GGTGTTACGTTTAGTTACGGAATAATGAGAGAAG 112G00-455-244 Marker ATTG sWSS1218 AFM309yf1 D7S680 Genetic GCTCAACTGACAGAAAACGACTATGTAAAAGAAA 154 G00-307-733 Marker TGCC sWSS1402D7S1916 YAC End AAAGGGCTTCTAATCTACCCTTCCAACTTCTTTG 137 G00-344-044 ACsWSS999 D7S1674 YAC  TAAACCCCCTTTCTGTTCTTGCATAATAGTCACA 105 G00-334-839Insert CCC sWSS1751 D7S2186 YAC End CCAAAATCAGAATTGTCAGAAGAAACCGAAGTTC186 G00-455-238 AGATACAG sWSS1174 AFM218xf10 D7S514 Genetic AATATCTGACATTGGCACTTAGACCTGAGAAAAG 144 G00-307-700 Marker AG sWSS2061D7S2184 YAC End GTTGCACAATACAAAATCCCTTCCATTAGTGTCT 200 G00-455-241 TATAGsWSS2588 D7S2187 YAC End ATCACTACACACCTAATCCCATTCTACATTTCCA 117G00-455-253 CC sWSS808 PAX4 PAX4 Gene GGCTGTGTGAGCAAGATCCTAGGATTGCCAGGCA153 G00-455-2 AAGAGGGCTGGAC sWSS1392 AFM206xc1 D7S635 Genetic CTCAGGTATGTCTTTATCTGTCTCTGCATTCTTT 75 G00-307-815 Marker TC sWSS1148AFM199xh12 D7S504 Genetic  GACACATACAAACACAAGATTGAGTTGAGTGTAG 60G00-307-652 Marker TAG sWSS1529 D7S1943 YAC EndCAGGGATTTCTAATTGTCAAAAGATGGAGGCTTT 116 G00-334-119 TG sWSS2619 OB OBGene CGTTAAGGGAAGGAACTCTGGTGGCTTAGAGGAG 106 G00-455-256 TCAGGGA sWSS404D7S1956 Lambda  ACCAGGGTCAATACAAAGTAATGTGTCCTTCTTG 122 G00-334-251 CloneCC sWSS2367 AFMa345wc9 D7S1875 Genetic CAATCCTGGCTTCATTTGAAGGTGGGTAGGATGC 81 G00-455-250 Marker TA

TABLE 4Microsatellite markers in the YAC contig containing the human OB geneThe 8 microsatellite markers mapped to the YAC contig containing the humanOB gene (FIG. 4) are listed. In each case, the marker name (indicated as thealias in Table 3), type of microsatellite motif [tetranucleotide (Tetra) repeator (CA)n repeat], GDB-assigned locus name, primer sequences utilized for PCR-basedgenotype analysis, and GDB identification number are indicated. Additionaldetails regarding the PCR assays and the polymorphisms are available in GDB.Marker Name Type Locus Primers GDB ID No. UT528 Tetra. D7S1498TGCAGTAAGCTGTGATTGAGGTGCAGCTTTAATTGTGAGC G00-312-446 AFMa065zg9 (CA)nD7S1873 AGCTTCAAGACTTTNAGCCTGGTCAGCAGCACTGTGATT G00-437-253 AFMa125wh1(CA)n D7S1874 TCACCTTGAGATTCCATCCAACACCGTGGTCTTATCAAA G00-437-263AFM309yf10 (CA)n D7S680 CATCCAAGTTGGCAGTTTTTAGATGCTGAATTCCCAGACAG00-200-283 AFM218xf10 (CA)n D7S514 TGGGCAACACAGCAAATGCAGTTAGTGCCAATGTCAG00-188-404 AFM206xc1 (CA)n D7S635 CCAGGCCATGTGGAACAGTTCTTGGCTTGCGTCAGTG00-199-240 AFM199xh12 (CA)n D7S504 TCTGATTGCTGGCTGCGCGCGTGTGTATGTGAGG00-188-280 AFMa345wc9 (CA)n D7S1875AGCTCTTGGCAAACTCACATGCCTAAGGGAATGAGACACA G00-437-259

The human OB-specific STS (sWSS2619) was designed using DNA sequenceobtained from the 3 untranslated region of the cDNA. The humanPAX4-specific STS (sWSS808) was developed using the following strategy.Oligonucleotide primers specific for the mouse Pax4 gene[GGCTGTGTGAGCAAGATCCTAGGA and GGGAGCCTTGTCCTGGGTACAAAG (Walther et al.1991)] were used to amplify a 204-bp fragment from human genomic DNA(which was the same size product as that generated from mouse genomicDNA). This PCR assay was not suitable for identifying correspondingYACs, since a similarly-sized (200-bp) product was also amplified fromyeast DNA. However, DNA sequence analysis of the PCR product generatedfrom human DNA revealed substitutions at 20 positions among the 156bases analyzed (data not shown). Using this human-specific sequence, anew primer (TTGCCAGGCAAAGAGGGCTGGAC) was designed and used with thefirst of the above mouse Pax4-specific primers (see Table 3). Theresulting human PAX4-specific PCR assay did not amplify a significantproduct from yeast DNA and was thus used for identifying correspondingYACs.

Identification of YACs by PCR-Based Screening

Most of the YACs depicted in FIG. 34 were derived from a collection ofclones highly enriched for human chromosome 7 DNA [the chromosome 7 YACresource (Green et al. 1995a)] using a PCR-based screening strategy(Green et al. 1995a; Green and Olson, 1990). In a few cases, clones wereisolated by PCR-based screening (Green and Olson, 1990) of availabletotal human genomic YAC libraries constructed at CEPH (Dausset et al.1992; Albertsen et al. 1990) or ICI (Anand et al. 1990; Anand et al.1989). Each YAC is named using the prefix yWSS followed by a uniquenumber.

Results and Discussion

Examination of the tissue expression of the human OB gene by northernblot analysis revealed that OB RNA is expressed at a high level in humanadipose tissue and much lower levels in placenta and heart (FIG. 33).The size of the RNA (˜4.5 kb) was equivalent in human and mouse as wellas in each of the expressing tissues. In these studies, five-fold highersignals were seen in 10 μg of total adipose tissue RNA as in 2 μg ofpolyA+ placental RNA. A five-fold lower signal was seen in polyA+ RNAfrom heart compared to placenta. It is estimated that the level of OBRNA is ˜250-fold lower in placenta than in adipose tissue. In thisexperiment, OB RNA as not detected in any of the other tissues analyzed,including brain, lung, liver, skeletal muscle, kidney, and pancreas.Additional experiments did not reveal OB RNA in spleen, thymus,prostate, testis, ovary, small intestine, colon, peripheral bloodleukocytes, or in fetal brain, liver, or kidneys (data not shown). It ispossible that OB is expressed at an undetectable level (by northern blotanalysis) in these latter tissues or in other tissues that were notstudied. The observed pattern of expression in human differs somewhatfrom mouse, in which ob RNA is detected almost exclusively in adiposetissue.

Comparative Mapping of the Ob Gene Region in the Mouse and Human Genomes

The mouse ob gene is located on proximal chromosome 6 in a regionhomologous with a portion of human chromosome 7q. Genes within thissegment include (from proximal to distal): Met protooncogene, the cysticfibrosis transmembrane conductance regulator (Cftr), pairedbox-containing gene 4 (Pax4), ob, and carboxypeptidase A (Cpa) (Zhang etal. 1994; Friedman et al. 1991). In mouse, genetic mapping was used todemonstrate that Pax4 is tightly linked to ob (Walther et al. 1991;Zhang et al. 1994). The physical distance between ob and Pax4 was foundto be ˜1 megabase pairs (Mb) (Zhang et al. 1994). Based on thesecomparative mapping studies, it was expected that the human OB genewould reside between PAX4 and CPA on chromosome 7q. Furthermore, sincehuman CFTR (Heng et al. 1993) and PAX4 (Tamura et al. 1994) were mappedby fluorescence in situ hybridization (FISH) to 7q31.3 and 7q32,respectively, the most likely cytogenetic position of the human OB genewould be in the vicinity of the 7q31.3-q32 boundary.

Mapping the OB Gene on Human Chromosome 7

An STS (sWSS2619) amplifying a small segment of the 3 untranslatedregion of the human OB gene was used to screen a collection of YACclones that is highly enriched for human chromosome 7 DNA (Green et al.1995a), and 9 YACs were identified (yWSS691, yWSS1332, yWSS1998,yWSS2087, yWSS3319, yWSS3512, yWSS4875, yWSS4970, and yWSS5004). Toverify that these YACs contain the authentic human OB gene, 2 additionalexperiments were performed. First, each of the YACs was tested with asecond human OB-specific PCR assay, and all were found to be positive(data not shown). Second, yeast DNA from each clone was digested withEcoRI and analyzed by gel-transfer hybridization using a human OBcDNA-derived probe. In all instances, a single hybridizing band wasseen, and this band was the same size in the YACs and a P1 clone knownto contain the human OB gene (data not shown).

Using the computer program SEGMAP (Green and Green, 1991a; C. L. Magnessand P. Green, unpublished data) and other YAC-based STS-content datathat we have generated for chromosome 7 (Green et al. 1991b; Green etal. 1994; Green et al. 1995a), the human OB gene was found to residewithin the YAC contig depicted in FIG. 2. Specifically, this contigconsists of 43 overlapping YACs and 19 uniquely-ordered STSs. Detailsabout each of the 19 STSs are provided in Table 3. In addition to theOB-specific STS, the contig also contains an STS (sWSS808) specific forthe human PAX4 gene (Tamura et al. 1994; Stapleton et al. 1993), 7 STSsderived from chromosome 7-specific YACs, 2 STSs derived from chromosome7-specific lambda clones, and, importantly, 8 microsatellite-specificSTSs. Additional details about these 8 genetic markers, includingsequences of the primers used for genotype analysis, are provided inTable 4. Of note, there is redundant YAC-based connectivity throughoutthe contig (i.e., there are 2 or more YACs connecting each adjacent pairof STSs), lending strong support for the relative order of STSs shown inFIG. 34.

As depicted in FIG. 34, the predicted orientation of the humanOB-containing YAC contig is such that sWSS1734 is the centromeric-mostSTS (i.e., closest to CFTR) while sWSS2367 is the telomeric-most STS(i.e., closest to CPA). This orientation is predominantly based oncomparative mapping data, which places Pax4 proximal and ob distalwithin the syntenic block present in mouse and human DNA (Zhang et al.1994). The OB gene maps near the telomeric end of the contig, based onthe placement of the OB-specific STS (sWSS2619).

While the contig shown in FIG. 34 was deduced by SEGMAP withoutconsideration of YAC sizes (thereby displaying STSs equidistant from oneanother), a similar analysis of the data by SEGMAP that accounted forYAC sizes indicated that the total size of the region covered by thecontig is just over 2 Mb (data not shown). Thus, while all 8 of themicrosatellite-specific STSs (Table 4) are contained within a genomicinterval spanning roughly 2 Mb, the 3 closest to the telomeric end ofthe contig (sWSS1392, sWSS1148, and sWSS2367) are particularly close tothe OB gene itself (perhaps within an interval as small as ˜500 kb). Infact, all 3 of the latter STSs are present in at least 1 of the humanOB-containing YACs. Of note, the interval between human PAX4 (sWSS808)and OB (sWSS2619) is estimated to be ˜400 kb, whereas this region waspredicted to span ˜1 Mb in mouse (Zhang et al. 1994). Finally, 3 of theYACs within the contig (yWSS691, yWSS999, and yWSS2935) have also beenanalyzed by FISH, and each was found to hybridize exclusively to 7q31.3(T. Featherstone and E. D. Green, unpublished data). One of these YACs(yWSS691) contains the OB-specific STS, while the other 2 clones containthe PAX4-specific STS. The latter results are generally consistent withthe previous cytogenetic assignment of human PAX4 to 7q32 (Tamura et al.1994). Based on these data, the human OB gene can be assigned tocytogenetic band 7q31.3.

REFERENCES FOR THIS EXAMPLE

-   Albertsen, H. M., H. Abderrahim, H. M. Cann, J. Dausset, D. Le    Paslier, and D. Cohen. 1990. Construction and characterization of a    yeast artificial chromosome library containing seven haploid genome    equivalents. Proc. Natl. Acad. Sci. USA 87: 4256-4260.-   Anand, R., A. Villasante, and C. Tyler-Smith. 1989. Construction of    yeast artificial chromosome libraries with large inserts using    fractionation by pulsed-field gel electrophoresis. Nucl. Acids Res.    17: 3425-3433.-   Anand, R, J. H. Riley, R. Butler, J. C. Smith, and A. F.    Markham. 1990. A 3.5 genome equivalent multi access YAC library:    construction, characterisation, screening and storage. Nucl. Acids    Res. 18; 1951-1956.-   Ballabio, A. 1993. The rise and fall of positional cloning? Nature    Genet. 3: 277-279.-   Chirgwin, J. J., A. E. Przbyla, R. J. MacDonald, and W. J.    Rutter. 1979. Isolation of biologically active ribonucleic acid from    sources enriched in ribonuclease. Biochem. 18: 5294-5299.-   Coleman, D. L. 1978. Obese and diabetes: two mutant genes causing    diabetes-obesity syndromes in mice. Diabetologia 14: 141-148.-   Collins, F. S. 1992. Positional cloning: let's not call it reverse    anymore. Nature Genet. 1: 3-6.-   Collins, F. and D. Galas. 1993. A new five-year plan for the U.S.    human genome project. Science 262: 43-46.-   Collins, F. S. 1995. Positional cloning moves from perditional to    traditional. Nature Genetics 9: 347-350.-   Dausset, J., P. Ougen, H. Abderrahim, A. Billault, J.-L. Sambucy, D.    Cohen, and D. Le Paslier. 1992. The CEPH YAC library. Behring Inst.    Mitt. 91: 13-20.-   Friedman, J. M., R. L. Leibel, D. S. Siegel, J. Walsh, and N.    Bahary. 1991. Molecular mapping of the mouse ob mutation. Genomics    11: 1054-1062.-   Green, E. D. and M. V. Olson. 1990. Systematic screening of yeast    artificial-chromosome libraries by use of the polymerase chain    reaction. Proc. Natl. Acad. Sci. USA 87: 1213-1217.-   Green, E. D. and P. Green. 1991a. Sequence-tagged site (STS) content    mapping of human chromosomes: theoretical considerations and early    experiences. PCR Methods Applic. 1: 77-90.-   Green, E. D., R. M. Mohr, J. R. Idol, M. Jones, J. M.    Buckingham, L. L. Deaven, R. K. Moyzis, and M. V. Olson. 1991b.    Systematic generation of sequence-tagged sites for physical mapping    of human chromosomes: application to the mapping of human chromosome    7 using yeast artificial chromosomes. Genomics 11: 548-564.-   Green, E. D. 1993. Physical mapping of human chromosomes: generation    of chromosome-specific sequence-tagged sites. In Methods in    Molecular Genetics (Vol. 1): Gene and Chromosome Analysis (Part A).    (ed. K. W. Adolph), pp. 192-210. Academic Press, Inc. San Diego.-   Green, E. D., J. R. Idol, R. M. Mohr-Tidwell, V. V. Braden, D. C.    Peluso, R. S. Fulton, H. F. Massa, C. L. Magness, A. M. Wilson, J.    Kimura, J. Weissenbach, and B. J. Trask. 1994. Integration of    physical, genetic and cytogenetic maps of human chromosome 7:    isolation and analysis of yeast artificial chromosome clones for 117    mapped genetic markers. Hum. Mol. Genet. 3: 489-501.-   Green, E. D., V. V. Braden, R. S. Fulton, R. Lim, M. S.    Ueltzen, D. C. Peluso, R. M. Mohr-Tidwell, J. R. Idol, L. M.    Smith, I. Chumakov, D. Le Paslier, D. Cohen, T. Featherstone, and P.    Green. 1995a. A human chromosome 7 yeast artificial chromosome (YAC)    resource: construction, characterization, and screening. Genomics    25: 170-183.-   Green, E. D., D. R. Cox, and R. M. Myers. 1995b. The human genome    project and its impact on the study of human disease. In The    metabolic and Molecular Bases of Inherited Disease. (eds. C. R.    Scriver, A. L. Beaudet, W. S. Sly, and D. Valle), pp. 401-436.    McGraw Hill, New York.-   Gyapay, G., J. Morissette, A. Vignal, C. Dib, C. Fizames, P.    Millasseau, S. Marc, G. Bernardi, M. Lathrop, and J.    Weissenbach. 1994. The 1993-94 Genethon human genetic linkage map.    Nature Genet. 7: 246-249.-   Heng, H. H. Q., X.-M. Shi, and L.-C. Tsui. 1993. Fluorescence in    situ hybridization mapping of the cystic fibrosis transmembrane    conductance regulator (CFTR) gene to 7q31.3. Cytogenet. Cell Genet.    62: 108-109.-   Hillier, L. and P. Green. 1991. OSP: A computer program for choosing    PCR and DNA sequencing primers. PCR Methods Applic. 1: 124-128.-   Olson, M. V. 1993. The human genome project. Proc. Natl. Acad. Sci.    USA 90: 4338-4344.-   Stapleton, P., A. Weith, P. Urbanek, Z. Kozmik, and M.    Busslinger. 1993. Chromosomal localization of seven PAX genes and    cloning of a novel family members, PAX-9. Nature Genet. 3: 292-298.-   Tamura, T., Y. Izumikawa, T. Kishino, H. Soejima, Y. Jinno, and N.    Niikawa. 1994. Assignment of the human PAX4 gene to chromosome band    7q32 by fluorescence in situ hybridization. Cytogenet. Cell Genet.    66: 132-134.-   Walther, C., J.-L. Guenet, D. Simon, U. Deutsch, B. Jostes, M. D.    Goulding, D. Plachov, R. Balling, and P. Gros. 1991. Pax: a murine    multigene family of paired box-containing genes. Genomics 11:    424-434.-   Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold, and J. M.    Friedman. 1994. Positional cloning of the mouse obese gene and its    human homologue. Nature 372: 425-432.

Example Human Ob Peptide is Biologically Active in Mice

Groups of 10 ob/ob mice were treated by i.p. injection with 10 μg/g/dayrecombinant (bacterial) human and murine ob peptide or saline. Afterfour days, the group receiving saline gained 0.3 g. The group receivingmurine ob lost 3.2 g. The group receiving human ob lost 2 g (p<0.01compared to saline controls). These groups were also tested for foodintake. The data are shown in Table 5.

TABLE 5 Food intake/day (g) of treated ob/ob mice Treatment Day 0 Day 1Day 2 Day 3 saline 13 13 12.9 13.2 murine ob 14 3 4 4.6 human ob 14.210.2 8.7 7.7

These data demonstrate that human ob is biologically active in mice.

Example A High Dose of Ob Affects Wild-Type Mice

Wild type mice (C57Bl6J+/?) were treated with 10 μg/g/day i.p. ofrecombinant murine ob, and body mass measured every four days. Theresults are shown in Table 6.

TABLE 6 Body mass of normal mice receiving ob Treatment Day 0 Day 4 Day8 Day 12 Day 16 saline 22 g   22 g 22.5 g   23 g 22.5 g murine ob 2220.5 20.7 20.8 21.8

These data demonstrate that ob affects the body mass of wild-type aswell as obese (ob/ob) mice, albeit to a much smaller degree.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

1. An isolated DNA molecule comprising a DNA sequence selected from thegroup consisting of: A. SEQ ID NO:1; B. SEQ ID NO:3; C. SEQ ID NO:22;and D. a DNA sequence encoding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:23,or biologically active fragment thereof.